Organism identification panel

ABSTRACT

Methods and containers are provided for identifying a species, illustratively a bacterial species. Illustrative methods comprise amplifying various genes in the nucleic acid from the bacterial species in a single reaction mixture using pairs of outer first-stage primers designed to hybridize to generally conserved regions of the respective genes to generate a plurality of first-stage amplicons, dividing the reaction mixture into a plurality of second-stage reactions, each using a unique pair of second-stage primers, each pair of second-stage primers specific for a target bacterial species or subset of bacterial species, detecting which of the second-stage reactions amplified, and identifying the bacterial species based on second-stage amplification. Methods for determining antibiotic resistance are also provided, such methods also using first-stage primers for amplifying genes known to affect antibiotic resistance a plurality of the second-stage reactions wherein each pair of second-stage primers specific for a specific gene for conferring antibiotic resistance.

PRIORITY STATEMENT

This application is a continuation application of, and claims priorityto, U.S. application Ser. No. 12/594,478, filed Mar. 5, 2014 (allowed),which is a 35 U.S.C. §371 national phase application of PCT ApplicationSerial No. PCT/US2008/058993, filed Apr. 1, 2008, which claims thebenefit, under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationSer. No. 60/921,342, filed Apr. 2, 2007, the entire contents of each ofwhich are incorporated by referenced herein.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant Nos. U01AI061611, K12 HD001410, and R43 AI063695 awarded by National Institutesof Health. The government has certain rights in the invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 1267-4TSCT_ST25.txt, 14,080 bytes in size, generated onJul. 25, 2017 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosure.

BACKGROUND OF THE INVENTION

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. A challengeto using PCR as a primary means of diagnosis is the variety of possiblecausative organisms and the low levels of organism present in somepathological specimens. It is often impractical to run large panels ofPCR assays, one for each possible causative organism, most of which areexpected to be negative. The problem is exacerbated when pathogennucleic acid is at low concentration and requires a large volume ofsample to gather adequate reaction templates. In some cases there isinadequate sample to assay for all possible etiological agents. Asolution is to run “multiplex PCR” wherein the sample is concurrentlyassayed for multiple targets in a single reaction. While multiplex PCRhas proved to be valuable in some systems, shortcomings exist concerningrobustness of high level multiplex reactions and difficulties for clearanalysis of multiple products. To solve these problems, the assay may besubsequently divided into multiple secondary PCRs. Nesting secondaryreactions within the primary product increase robustness. However, thisfurther handling can be expensive and may lead to contamination or otherproblems.

Similarly, immuno-PCR (“iPCR”) has the potential for sensitive detectionof a wide variety of antigens. However, because traditional ELISAtechniques have been applied to iPCR, iPCR often suffers fromcontamination issues that are problematic using a PCR-based detectionmethod.

The present invention addresses various issues of contamination inbiological analysis.

SUMMARY OF THE INVENTION

Accordingly, a rapid, sensitive assay that simultaneously assays formultiple biological substances, including organisms, is provided. Theself-contained system illustratively employs an inexpensive disposableplastic pouch in a self-contained format, allowing for nested PCR andother means to identify bio-molecules, illustratively while minimizingcontamination and providing for robust amplification.

In one aspect of the invention, methods for identifying an organism areprovided, the methods comprising the steps of amplifying, in a singlereaction mixture containing nucleic acid from the organism, a pluralityof conserved genes using outer first-stage primers designed to hybridizeto generally conserved regions of the respective genes to generate aplurality of first-stage amplicons, dividing the reaction mixture into aplurality of second-stage reactions, subjecting each of a plurality ofthe second-stage reactions to amplification conditions, wherein each ofthe plurality of second-stage reactions uses a unique pair ofsecond-stage primers, each pair of second-stage primers specific for aspecific organism or genus of organisms, detecting which of thesecond-stage reactions amplified, and identifying the bacterial speciesbased on amplification of the second-stage reactions. Illustratively,the steps of amplifying, dividing, subjecting, detecting, andidentifying all take place in a single closed container. In a furtherillustrative embodiment, the step of extracting the nucleic acid alsotakes place in the closed container.

In the illustrative embodiments, the organism is a bacterial species.Thus, in another aspect of the invention, methods are provided foridentifying a bacterial species, the methods comprising the steps ofobtaining a sample containing the bacterial species, amplifying, in asingle reaction mixture containing nucleic acid from the sample, aplurality of bacterial genes using pairs of outer first-stage primersdesigned to hybridize to generally conserved regions of the respectivegenes to generate a plurality of first-stage amplicons, dividing thereaction mixture into a plurality of second-stage reactions, subjectingeach of a plurality of the second-stage reactions to amplificationconditions, wherein each of the plurality of second-stage reactions usesa unique pair of second-stage primers, each pair of second-stage primersspecific for a target bacterial species or group of bacterial species,detecting which of the second-stage reactions amplified, and identifyingthe bacterial species based on which of the second-stage reactionsamplified.

In another aspect of this invention methods are provided for determiningantibiotic resistance in a sample, the methods comprising the steps ofamplifying, in a single reaction mixture containing nucleic acid fromthe organism, a plurality of genes that confer antibiotic resistanceusing outer first-stage primers designed to hybridize to regions of therespective genes to generate a plurality of first-stage amplicons,dividing the reaction mixture into a plurality of second-stagereactions, subjecting each of a plurality of the second-stage reactionsto amplification conditions, wherein each of the plurality ofsecond-stage reactions uses a unique pair of second-stage primers, eachpair of second-stage primers specific for a specific gene for conferringantibiotic resistance, detecting which of the second-stage reactionsamplified, and identifying the organisms based on amplification of thesecond-stage reactions. In such methods, species identification may ormay not be performed.

In yet another aspect of this invention, containers are provided foridentifying a species using two-stage nucleic acid amplification on asample in a closed system, the comprising a first-stage reaction zonecomprising a plurality of outer first-stage primers for amplifying aplurality of genes of the species, the outer first-stage primersdesigned to hybridize to generally conserved regions of the respectivegenes, the first-stage reaction zone configured for first-stageamplification of the sample, and a second-stage reaction zone fluidlyconnected to the first-stage reaction zone, the second-stage reactionzone comprising a plurality of second-stage reaction chambers, eachsecond-stage reaction chamber comprising a pair of primers specific fora species or group of species.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flexible pouch according to one embodiment of thisinvention.

FIG. 2 shows an embodiment of the cell lysis zone of the flexible pouchaccording to FIG. 1.

FIG. 2a shows an embodiment of a portion of a bladder corresponding tothe cell lysis zone shown in FIG. 2.

FIG. 2b shows an embodiment of the cell lysis zone of the flexible pouchaccording to FIG. 1 having an alternative vortexing mechanism.

FIG. 3 shows an embodiment of the nucleic acid preparation zone of theflexible pouch according to FIG. 1.

FIG. 4 shows an embodiment of the first-stage amplification zone of theflexible pouch according to FIG. 1.

FIG. 5 is similar to FIG. 1, except showing an alternative embodiment ofa pouch.

FIG. 5a is a cross-sectional view of the fitment of the pouch of FIG. 5.

FIG. 5b is an enlargement of a portion of the pouch of FIG. 5.

FIG. 6 is a perspective view of another alternative embodiment of apouch.

FIG. 6a is a cross-sectional view of the fitment of the pouch of FIG. 6.

FIG. 7 shows illustrative bladder components for use with the pouch ofFIG. 6.

FIG. 8 is an exploded perspective view of an instrument for use with thepouch of FIG. 6, including the pouch of FIG. 6.

FIG. 9 shows a partial cross-sectional view of the instrument of FIG. 8,including the bladder components of FIG. 7, with the pouch of FIG. 6shown in shadow.

FIG. 10 shows a partial cross-sectional view of the instrument of FIG.8, including various bladders for pinch valves and the pouch of FIG. 6.

FIG. 11 shows schemes for ELISA and immuno-PCR, secondary antibody (A);capture antibody (C); enzyme (E); reporter antibody (R); bi-functionalbinding moiety (S) and antigen (T).

FIG. 12 is similar to FIG. 6, except showing a pouch configured forimmuno-PCR.

FIG. 13 is similar to FIG. 6, except showing a pouch configured for bothPCR and immuno-PCR.

FIG. 14 shows amplification curves from second-stage amplification of asample that was lysed and amplified in a pouch of FIG. 5 ( - - -positive control;

S. cerevisiae target 1; - S. cerevisiae target 2; - S. cerevisiae target3;

S. pombe target 1;

S. pombe target 2; - - - - negative controls).

FIG. 15 is similar to FIG. 6, except showing a pouch having asecond-stage high density array.

FIG. 15a shows a modification of a component of the instrument of FIG.8. A support member has been provided with a motor configured for usewith the pouch of FIG. 15.

FIG. 16 is an exploded perspective view of the second-stage high densityarray of FIG. 15.

FIG. 17 is a bottom view of the second-stage high density array of FIG.15, shown during construction of the second-stage high density array.

FIGS. 18A-18C show identification of Gram-negative organisms using rpoBand gyrB. Outer first-stage amplicons were generated from 5 organisms(P. aeruginosa (Pa, light green), E. coli (Ec, red), K. pneumoniae (Kp,purple), K. oxytoca (Ko, pink) and H. influenzae (Hi, blue)) withdegenerate outer primers targeting the rpoB and gyrB genes. Outerfirst-stage amplicons were nested into a set of organism-specific innersecond-stage primers with the resulting real time amplification curvesshown.

FIG. 18A shows that second-stage primers specific for either the rpoB orgyrB gene of Pa amplify only from the Pa template, except for a minoramplification with Ec in late cycles.

FIG. 18B shows that the rpoB “enteric” second-stage primer amplifies Ec,Kp and Ko, but for the gyrB gene a specific primer targets only Kp.

FIG. 18C shows that second-stage primers specific for either the rpoB orgyrB gene of Hi amplify only from the Hi template, except for a minoramplification with Ko near the end of the reaction.

FIGS. 19A-19D show Identification of Gram-positive organisms using rpoB.Outer first-stage amplicons were generated from 4 organisms (S.pneumoniae (Sp, blue), S. agalactiae (Sag, green), S. aureus (Sa, red),and L. monocytogenes (Lm, orange)) with degenerate outer first-stageprimers targeting the rpoB gene. Outer first-stage amplicons were nestedinto a set of organism-specific inner primers with the resulting realtime amplification curves shown. Second-stage primers specific for therpoB gene of Sp (FIG. 19A), Sag (FIG. 19B), Sa (FIG. 19C), or Lm (FIG.19D) have significant amplification curves only from the appropriatetemplates. Minor amplification curves with other templates are underinvestigation and likely result from the formation of “primer dimers”(especially in the case of the S. aureus primers) or possiblycross-amplification.

FIGS. 20A-20B show melting profiles of amplicons from the amplificationreactions shown in FIG. 18. The melting profile of each amplicon wasgenerated using the dye LCGreen® Plus Amplicons melted withcharacteristic Tms that ranged from 78° C. for H. influenzae to 90° C.for P. aeruginosa.

FIGS. 21a-21b . FIG. 21a shows results for N. meningitidis. FIG. 21ashows amplification of the gyrB gene, while FIG. 21b shows the resultsfor the rpoB gene for this target.

FIGS. 22a-22d show differentiation of enteric organisms usingpreferential primers in rpoB amplification. FIG. 22a shows amplificationusing the “pan-enteric” primers, FIG. 22b shoes amplification using E.coli preferential primers, FIG. 22c shows amplification using K.pneumoniae primers, and FIG. 22d shows amplification using E. cloacaeprimers.

FIGS. 23a-23b show a decision tree for enteric identification based onCp fingerprint. FIG. 23a is a table showing illustrative Cp cut-offvalues, and FIG. 23b shows a flow-chart using the values of FIG. 23 a.

DETAILED DESCRIPTION

The self-contained nucleic acid analysis pouches described herein may beused to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. In one embodiment, the pouchis used to assay for multiple pathogens. Illustratively, various stepsmay be performed in the optionally disposable pouch, including nucleicacid preparation, primary large volume multiplex PCR, dilution ofprimary amplification product, and secondary PCR, culminating withreal-time detection and/or post-amplification analysis such asmelting-curve analysis. It is understood, however, that pathogendetection is one exemplary use and the pouches may be used for othernucleic acid analysis or detection of other substances, including butnot limited to peptides, toxins, and small molecules. Further, it isunderstood that while the various steps may be performed in pouches ofthe present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependant amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. It is understood that protocolsmay need to be adjusted accordingly.

FIG. 1 shows an illustrative self-contained nucleic acid analysis pouch10. Pouch 10 has a cell lysis zone 20, a nucleic acid preparation zone40, a first-stage amplification zone 60, and a second-stageamplification zone 80. A sample containing nucleic acid is introducedinto the pouch 10 via sample injection port 12. Pouch 10 comprises avariety of channels and blisters of various sizes and is arranged suchthat the sample flows through the system. The sample passes through thevarious zones and is processed accordingly.

Sample processing occurs in various blisters located within pouch 10.Various channels are provided to move the sample within and betweenprocessing zones, while other channels are provided to deliver fluidsand reagents to the sample or to remove such fluids and reagents fromthe sample. Liquid within pouch 10 illustratively is moved betweenblisters by pressure, illustratively pneumatic pressure, as describedbelow, although other methods of moving material within the pouch arecontemplated.

While other containers may be used, illustratively, pouch 10 is formedof two layers of a flexible plastic film or other flexible material suchas polyester, polyethylene terephthalate (PET), polycarbonate,polypropylene, polymethylmethacrylate, and mixtures thereof that can bemade by any process known in the art, including extrusion, plasmadeposition, and lamination. Metal foils or plastics with aluminumlamination also may be used. Other barrier materials are known in theart that can be sealed together to form the blisters and channels. Ifplastic film is used, the layers may be bonded together, illustrativelyby heat sealing. Illustratively, the material has low nucleic acidbinding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by tryingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in the blisters 82 of the second stageamplification zone 80 of pouch 10, then one or both layers at blisters82 would be left without the foil. In the example of PCR, film laminatescomposed of polyester (Mylar, Dupont, Wilmington Del.) of about 0.0048inch (0.1219 mm) thick and polypropylene films of 0.001-0.003 inch(0.025-0.076 mm) thick perform well. Illustratively, pouch 10 is made ofa clear material capable of transmitting approximately 80%-90% ofincident light.

In the illustrative embodiment, the materials are moved between blistersby the application of pressure, illustratively pneumatic pressure, uponthe blisters and channels. Accordingly, in embodiments employingpneumatic pressure, the pouch material illustratively is flexible enoughto allow the pneumatic pressure to have the desired effect. The term“flexible” is herein used to describe a physical characteristic of thematerial of pouch. The term “flexible” is herein defined as readilydeformable by the levels of pneumatic pressure used herein withoutcracking, breaking, crazing, or the like. For example, thin plasticsheets, such as Saran™ wrap and Ziploc® bags, as well as thin metalfoil, such as aluminum foil, are flexible. However, only certain regionsof the blisters and channels need be flexible; even in embodimentsemploying pneumatic pressure. Further, only one side of the blisters andchannels need to be flexible, as long as the blisters and channels arereadily deformable. Other regions of the pouch 10 may be made of a rigidmaterial or may be reinforced with a rigid material.

Illustratively, a plastic film is used for pouch 10. A sheet of metal,illustratively aluminum, or other suitable material, may be milled orotherwise cut, to create a die having a pattern of raised surfaces. Whenfitted into a pneumatic press (illustratively A-5302-PDS, JanesvilleTool Inc., Milton Wis.), illustratively regulated at an operatingtemperature of 195° C., the pneumatic press works like a printing press,melting the sealing surfaces of plastic film only where the die contactsthe film. Various components, such as PCR primers (illustrativelyspotted onto the film and dried), antigen binding substrates, magneticbeads, and zirconium silicate beads may be sealed inside variousblisters as the pouch 10 is formed. Reagents for sample processing canbe spotted onto the film prior to sealing, either collectively orseparately. In one embodiment, nucleotide tri-phosphates (NTPs) arespotted onto the film separately from polymerase and primers,essentially eliminating activity of the polymerase until the reaction ishydrated by an aqueous sample. If the aqueous sample has been heatedprior to hydration, this creates the conditions for a true hot-start PCRand reduces or eliminates the need for expensive chemical hot-startcomponents. This separate spotting is discussed further below, withrespect to FIG. 5b , but it is understood that such spotting may be usedwith any of the embodiments discussed herein.

When pneumatic pressure is used to move materials within pouch 10, inone embodiment a “bladder” may be employed. The bladder assembly 710, aportion of which is shown in FIG. 2a , may be manufactured in a processsimilar to that of making the pouch, but individual blisters in thebladder assembly 710 include pneumatic fittings (illustratively fitting724 a) allowing individual bladders within the bladder assembly 710 tobe pressurized by a compressed gas source. Because the bladder assemblyis subjected to compressed gas and may be used multiple times, thebladder assembly may be made from tougher or thicker material than thepouch. Alternatively, bladders may be formed from a series of platesfastened together with gaskets, seals, valves, and pistons. Otherarrangements are within the scope of this invention.

When pouch 10 is placed within the instrument, the pneumatic bladderassembly 710 is pressed against one face of the pouch 10, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 10. In addition to pneumaticbladders corresponding to many of the blisters of pouch 10, the bladderassembly may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch10. When activated, these additional pneumatic actuators form pinchvalves to pinch off and close the corresponding channels. To confineliquid within a particular blister of pouch 10, the pinch valvepneumatic actuators are inflated over the channels leading to and fromthe blister, such that the actuators function as pinch valves to pinchthe channels shut. Illustratively, to mix two volumes of liquid indifferent blisters, the pinch valve pneumatic actuator sealing theconnecting channel is depressurized, and the pneumatic bladders over theblisters are alternately pressurized, forcing the liquid back and forththrough the channel connecting the blisters to mix the liquid therein.The pinch valve pneumatic actuators may be of various shapes and sizesand may be configured to pinch off more than one channel at a time. Suchan illustrative pinch valve is illustrated in FIG. 1 as pinch valve 16,which may be used to close all injection ports. While pneumaticactuators are discussed herein, it is understood that other ways ofproviding pressure to the pouch are contemplated, including variouselectromechanical actuators such as linear stepper motors, motor-drivencams, rigid paddles driven by pneumatic, hydraulic or electromagneticforces, rollers, rocker-arms, and in some cases, cocked springs. Inaddition, there are a variety of methods of reversibly or irreversiblyclosing channels in addition to applying pressure normal to the axis ofthe channel. These include kinking the bag across the channel,heat-sealing, rolling an actuator, and a variety of physical valvessealed into the channel such as butterfly valves and ball valves.Additionally, small Peltier devices or other temperature regulators maybe placed adjacent the channels and set at a temperature sufficient tofreeze the fluid, effectively forming a seal. Also, while the design ofFIG. 1 is adapted for an automated instrument featuring actuatorelements positioned over each of the blisters and channels, it is alsocontemplated that the actuators could remain stationary, and the pouchcould be transitioned in one or two dimensions such that a small numberof actuators could be used for several of the processing stationsincluding sample disruption, nucleic-acid capture, first andsecond-stage PCR, and other applications of the pouch such asimmuno-assay and immuno-PCR. Rollers acting on channels and blisterscould prove particularly useful in a configuration in which the pouch istranslated between stations. Thus, while pneumatic actuators are used inthe presently disclosed embodiments, when the term “pneumatic actuator”is used herein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

With reference to FIG. 1, an illustrative sample pouch 10 configured fornucleic acid extraction and multiplex PCR is provided. The sample enterspouch 10 via sample injection port 12 in fitment 90. Injector port 12may be a frangible seal, a one-way valve, or other entry port. Vacuumfrom inside pouch 10 may be used to draw the sample into pouch 10, asyringe or other pressure may be used to force the sample into pouch 10,or other means of introducing the sample into pouch 10 via injector port12 may be used. The sample travels via channel 14 to the three-lobedblister 22 of the cell lysis zone 20, wherein cells in the sample arelysed. Once the sample enters three-lobed blister 22, pinch valve 16 isclosed. Along with pinch valve 36, which may have been already closed,the closure of pinch valve 16 seals the sample in three-lobed blister22. It is understood that cell lysis may not be necessary with everysample. For such samples, the cell lysis zone may be omitted or thesample may be moved directly to the next zone. However, with manysamples, cell lysis is needed. In one embodiment, bead-milling is usedto lyse the cells.

Bead-milling, by shaking or vortexing the sample in the presence oflysing particles such as zirconium silicate (ZS) beads 34, is aneffective method to form a lysate. It is understood that, as usedherein, terms such as “lyse,” “lysing,” and “lysate” are not limited torupturing cells, but that such terms include disruption of non-cellularparticles, such as viruses. FIG. 2 displays one embodiment of a celllysis zone 20, where convergent flow creates high velocity bead impacts,to create lysate. Illustratively, the two lower lobes 24, 26 ofthree-lobed blister 22 are connected via channel 30, and the upper lobe28 is connected to the lower lobes 24, 26 at the opposing side 31 ofchannel 30. FIG. 2a shows a counterpart portion of the bladder assembly710 that would be in contact with the cell lysis zone 20 of the pouch10. When pouch 10 is placed in an instrument, adjacent each lobe 24, 26,28 on pouch 10 is a corresponding pneumatic bladder 724, 726, 728 in thebladder assembly 710. It is understood that the term “adjacent,” whenreferring to the relationship between a blister or channel in a pouchand its corresponding pneumatic actuator, refers to the relationshipbetween the blister or channel and the corresponding pneumatic actuatorwhen the pouch is placed into the instrument. In one embodiment, thepneumatic fittings 724 a, 726 a of the two lower pneumatic bladders 724,726 adjacent lower lobes 24, 26 are plumbed together. The pneumaticfittings 724 a, 726 a and the pneumatic fitting 728 a of upper pneumaticbladder 728 adjacent upper lobe 28 are plumbed to the opposing side ofan electrically actuated valve configured to drive a double-actingpneumatic cylinder. Thus configured, pressure is alternated between theupper pneumatic bladder 728 and the two lower pneumatic bladders 724,726. When the valve is switched back and forth, liquid in pouch 10 isdriven between the lower lobes 24, 26 and the upper lobe 28 through anarrow nexus 32 in channel 30. As the two lower lobes 24, 26 arepressurized at the same time, the flow converges and shoots into theupper lobe 28. Depending on the geometry of the lobes, the collisionvelocity of beads 34 at the nexus 32 may be at least about 12 m/sec,providing high-impact collisions resulting in lysis. The illustrativethree-lobed system allows for good cell disruption and structuralrobustness, while minimizing size and pneumatic gas consumption. WhileZS beads are used as the lysing particles, it is understood that thischoice is illustrative only, and that other materials and particles ofother shapes may be used. It is also understood that otherconfigurations for cell lysis zone 20 are within the scope of thisinvention.

While a three-lobed blister is used for cell lysis, it is understoodthat other multi-lobed configurations are within the scope of thisinvention. For instance, a four-lobed blister, illustratively in acloverleaf pattern, could be used, wherein the opposite blisters arepressurized at the same time, forcing the lysing particles toward eachother, and then angling off to the other two lobes, which then may bepressurized together. Such a four-lobed blister would have the advantageof having high-velocity impacts in both directions. Further, it iscontemplated that single-lobed blisters may be used, wherein the lysingparticles are moved rapidly from one portion of the single-lobed blisterto the other. For example, pneumatic actuators may be used to close offareas of the single-lobed blister, temporarily forming a multi-lobedblister in the remaining areas. Other actuation methods may also be usedsuch as motor, pneumatic, hydraulic, or electromagnetically-drivenpaddles acting on the lobes of the device. Rollers or rotary paddles canbe used to drive fluid together at the nexus 32 of FIG. 2,illustratively if a recirculation means is provided between the upperand lower lobes and the actuator provides peristaltic pumping action.Other configurations are within the scope of this invention.

It may also be possible to move the sample and lysing particles quicklyenough to effect lysis within a single-lobed lysis blister withouttemporarily forming a multi-lobed blister. In one such alternativeembodiment, as shown in FIG. 2b , vortexing may be achieved by impactingthe pouch with rotating blades or paddles 21 attached to an electricmotor 19. The blades 21 may impact the pouch at the lysis blister or mayimpact the pouch near the lysis blister, illustratively at an edge 17adjacent the lysis blister. In such an embodiment, the lysis blister maycomprise one or more blisters. FIG. 15 shows an embodiment comprisingone such lysis blister 522. FIG. 15a shows a bead beating motor 19,comprising blades 21 that may be mounted on a first side 811 of secondsupport member 804, of instrument 800 shown in FIG. 8. It is understood,however, that motor 19 may be mounted on first support member 802 or onother structure of instrument 800.

FIG. 2a also shows pneumatic bladder 716 with pneumatic fitting 716 a,and pneumatic bladder 736 with pneumatic fitting 736 a. When the pouch10 is placed in contact with bladder assembly 710, bladder 716 lines upwith channel 12 to complete pinch valve 16. Similarly, bladder 736 linesup with channel 38 to complete pinch valve 36. Operation of pneumaticbladders 716 and 736 allow pinch valves 16 and 36 to be opened andclosed. While only the portion of bladder assembly 710 adjacent the celllysis zone is shown, it is understood that bladder assembly 710 would beprovided with similar arrangements of pneumatic blisters to control themovement of fluids throughout the remaining zones of pouch 10.

Other prior art instruments teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758 and 6,780,617, andco-pending U.S. patent application Ser. No. 10/478,453, hereinincorporated by reference. However, including the cell lysis within thesealed PCR vessel can improve ease of use and safety, particularly ifthe sample to be tested may contain a biohazard. In the embodimentsillustrated herein, the waste from cell lysis, as well as that from allother steps, remains within the sealed pouch. However, it is understoodthat the pouch contents could be removed for further testing.

Once the cells are lysed, pinch valve 36 is opened and the lysate ismoved through channel 38 to the nucleic acid preparation zone 40, asbest seen in FIG. 3, after which, pinch valve 36 is closed, sealing thesample in nucleic acid preparation zone 40. In the embodimentillustrated in FIG. 3, purification of nucleic acids takes thebead-milled material and uses affinity binding to silica-basedmagnetic-beads 56, washing the beads with ethanol, and eluting thenucleic acids with water or other fluid, to purify the nucleic acid fromthe cell lysate. The individual components needed for nucleic acidextraction illustratively reside in blisters 44, 46, 48, which areconnected by channels 45, 47, 49 to allow reagent mixing. The lysateenters blister 44 from channel 38. Blister 44 may be provided withmagnetic beads 56 and a suitable binding buffer, illustratively ahigh-salt buffer such as that of 1-2-3™ Sample Preparation Kit (IdahoTechnology, Salt Lake City, Utah) or either or both of these componentsmay be provided subsequently through one or more channels connected toblister 44. The nucleic acids are captured on beads 56, pinch valve 53is then opened, and the lysate and beads 56 may be mixed by gentlepressure alternately on blisters 44 and 58 and then moved to blister 58via pneumatic pressure illustratively provided by a correspondingpneumatic bladder on bladder assembly 710. The magnetic beads 56 arecaptured in blister 58 by a retractable magnet 50, which is located inthe instrument adjacent blister 58, and waste may be moved to a wastereservoir or may be returned to blister 44 by applying pressure toblister 58. Pinch valve 53 is then closed. The magnetic beads 56 arewashed with ethanol, isopropanol, or other organic or inorganic washsolution provided from blister 46, upon release of pinch valve 55.Optionally, magnet 50 may be retracted allowing the beads to be washedby providing alternate pressure on blisters 46 and 58. The beads 56 areonce again captured in blister 58 by magnet 50, and the non-nucleic acidportion of the lysate is washed from the beads 56 and may be moved backto blister 46 and secured by pinch valve 55 or may be washed away viaanother channel to a waste reservoir. Once the magnetic beads arewashed, pinch valve 57 is opened, releasing water (illustrativelybuffered water) or another nucleic acid eluant from blister 48. Onceagain, the magnet 50 may be retracted to allow maximum mixing of waterand beads 56, illustratively by providing alternate pressure on blisters48 and 58. The magnet 50 is once again deployed to collect beads 56.Pinch valve 59 is released and the eluted nucleic acid is moved viachannel 52 to first-stage amplification zone 60. Pinch valve 59 is thenclosed, thus securing the sample in first-stage amplification zone 60.

It is understood that the configuration for the nucleic acid preparationzone 40, as shown in FIG. 3 and described above, is illustrative only,and that various other configurations are possible within the scope ofthe present disclosure.

The ethanol, water, and other fluids used herein may be provided to theblisters in various ways. The fluids may be stored in the blisters, thenecks of which may be pinched off by various pinch valves or frangibleportions that may be opened at the proper time in the sample preparationsequence. Alternatively, fluid may be stored in reservoirs in the pouchas shown pouch 110 in FIG. 5, or in the fitment as discussed withrespect to pouch 210 of FIG. 6, and moved via channels, as necessary. Instill another embodiment, the fluids may be introduced from an externalsource, as shown in FIG. 1, especially with respect to ethanol injectionports 41, 88 and plungers 67, 68, 69. Illustratively, plungers 67, 68,69 may inserted into fitment 90, illustratively of a more rigidmaterial, and may provide a measured volume of fluid upon activation ofthe plunger, as in U.S. patent application Ser. No. 10/512,255, hereinincorporated by reference. The measured volume may be the same ordifferent for each of the plungers. Finally, in yet another embodiment,the pouch may be provided with a measured volume of the fluid that isstored in one or more blisters, wherein the fluid is contained withinthe blister, illustratively provided in a small sealed pouch within theblister, effectively forming a blister within the blister. At theappropriate time, the sealed pouch may then be ruptured, illustrativelyby pneumatic pressure, thereby releasing the fluid into the blister ofthe pouch. The instrument may also be configured the provide some or allof the reagents directly through liquid contacts between the instrumentand the fitment or pouch material provided that the passage of fluid istightly regulated by a one-way valve to prevent the instrument frombecoming contaminated during a run. Further, it will often be desirablefor the pouch or its fitment to be sealed after operation to prohibitcontaminating DNA to escape from the pouch. Various means are known toprovide reagents on demand such as syringe pumps, and to make temporaryfluid contact with the fitment or pouch, such as barbed fittings oro-ring seals. It is understood that any of these methods of introducingfluids to the appropriate blister may be used with any of theembodiments of the pouch as discussed herein, as may be dictated by theneeds of a particular application.

As discussed above, nested PCR involves target amplification performedin two stages. In the first-stage, targets are amplified, illustrativelyfrom genomic or reverse-transcribed template. The first-stageamplification may be terminated prior to plateau phase, if desired. Inthe secondary reaction, the first-stage amplicons may be diluted and asecondary amplification uses the same primers or illustratively usesnested primers hybridizing internally to the primers of the first-stageproduct. Advantages of nested PCR include: a) the initial reactionproduct forms a homogeneous and specific template assuring high fidelityin the secondary reaction, wherein even a relatively low-efficiencyfirst-stage reaction creates adequate template to support robustsecond-stage reaction; b) nonspecific products from the first-stagereaction do not significantly interfere with the second stage reaction,as different nested primers are used and the original amplificationtemplate (illustratively genomic DNA or reverse-transcription product)may be diluted to a degree that eliminates its significance in thesecondary amplification; and c) nested PCR enables higher-order reactionmultiplexing. First-stage reactions can include primers for severalunique amplification products. These products are then identified in thesecond-stage reactions. However, it is understood that first-stagemultiplex and second-stage singleplex is illustrative only and thatother configurations are possible. For example, the first-stage mayamplify a variety of different related amplicons using a single pair ofprimers, and second-stage may be used to target differences between theamplicons, illustratively using melting curve analysis.

Turning back to FIG. 1, the nucleic acid sample enters the first-stageamplification zone 60 via channel 52 and is delivered to blister 61. APCR mixture, including a polymerase (illustratively a Taq polymerase),dNTPs, and primers, illustratively a plurality of pairs of primers formultiplex amplification, may be provided in blister 61 or may beintroduced into blister 61 via various means, as discussed above.Alternatively, dried reagents may be spotted onto the location ofblister 61 upon assembly of pouch 10, and water or buffer may beintroduced to blister 61, illustratively via plunger 68, as shown inFIG. 1. As best seen in FIG. 4, the sample is now secured in blister 61by pinch valves 59 and 72, and is thermocycled between two or moretemperatures, illustratively by heat blocks or Peltier devices that arelocated in the instrument and configured to contact blister 61. However,it is understood that other means of heating and cooling the samplecontained within blister 61, as are known in the art, are within thescope of this invention. Non-limiting examples of alternativeheating/cooling devices for thermal cycling include having a air-cycledblister within the bladder, in which the air in the pneumatic blisteradjacent blister 61 is cycled between two or more temperatures; ormoving the sample to temperature zones within the blister 61,illustratively using a plurality of pneumatic presses, as in U.S. patentapplication Ser. No. 10/478,453, herein incorporated by reference, or bytranslating pouch 10 on an axis or providing pouch 10 with a rotarylayout and spinning pouch 10 to move the contents between heat zones offixed temperature.

Nucleic acids from pathogens are often co-isolated with considerablequantities of host nucleic acids. These host-derived nucleic acids ofteninteract with primers, resulting in amplification of undesired productsthat then scavenge primers, dNTPs, and polymerase activity, potentiallystarving a desired product of resources. Nucleic acids from pathogenicorganisms are generally of low abundance, and undesired product is apotential problem. The number of cycles in the first-stage reaction ofzone 60 may be optimized to maximize specific products and minimizenon-specific products. It is expected that the optimum number of cycleswill be between about 10 to about 30 cycles, illustratively betweenabout 15 to about 20 cycles, but it is understood that the number ofcycles may vary depending on the particular target, host, and primersequence.

Following the first-stage multiplex amplification, the first-stageamplification product is diluted, illustratively in incomplete PCRmaster mix, before fluidic transfer to secondary reaction sites.

FIG. 4 shows an illustrative embodiment for diluting the sample in threesteps. In the first step, pinch valve 72 is opened and the sampleundergoes a two-fold dilution by mixing the sample in blister 61 with anequal volume of water or buffer from blister 62, which is provided toblister 62, as well as blisters 64 and 66, as discussed above. Squeezingthe volume back and forth between blisters 61, 62 provides thoroughmixing. As above, mixing may be provided by pneumatic bladders providedin the bladder 710 and located adjacent blisters 61, 62. The pneumaticbladders may be alternately pressurized, forcing the liquid back andforth. During mixing, a pinch valve 74 prevents the flow of liquid intothe adjacent blisters. At the conclusion of mixing, a volume of thediluted sample is captured in region 70, and pinch valve 72 is closed,sealing the diluted sample in region 70. Pinch valve 74 is opened andthe sample is further diluted by water or buffer provided in either orboth of blisters 63, 64. As above, squeezing the volume back and forthbetween blisters 63, 64 provides mixing. Subsequently, pinch valve 74 isclosed, sealing a further diluted volume of sample in region 71. Finaldilution takes place illustratively by using buffer or water provided ineither or both of blisters 65, 66, with mixing as above. Illustrativelythis final dilution takes place using an incomplete PCR master mix(e.g., containing all PCR reagents except primers) as the fluid.Optional heating of the contents of blister 66 prior to second-stageamplification can provide the benefits of hot-start amplificationwithout the need for expensive antibodies or enzymes. It is understood,however, that water or other buffer may be used for the final dilution,with additional PCR components provided in second-stage amplificationzone 80. While the illustrative embodiment uses three dilution stages,it is understood that any number of dilution stages may be used, toprovide a suitable level of dilution. It is also understood that theamount of dilution can be controlled by adjusting the volume of thesample captured in regions 70 and 71, wherein the smaller the amount ofsample captured in regions 70 and 71, the greater the amount of dilutionor wherein additional aliquots captured in region 70 and/or region 71 byrepeatedly opening and closing pinch valves 72 and 74 and/or pinchvalves 74 and 76 may be used to decrease the amount of dilution. It isexpected that about 10⁻² to about 10⁻⁵ dilution would be suitable formany applications.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 10 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, and addition of adjuvants (such as DMSO, TMSO, orglycerol) optionally may be used to compensate for lower nucleic acidpurity. While purity issues are likely to be more of a concern withfirst-stage amplification, it is understood that similar adjustments maybe provided in the second-stage amplification as well.

While dilution and second-stage sample preparation are accomplished inthe illustrative embodiment by retaining a small amount of amplifiedsample in the blisters and channels of the first-stage PCR portion ofthe pouch, it is understood that these processes may also be performedin other ways. In one such illustrative example, pre-amplified samplecan be captured in a small cavity in a member, illustratively atranslating or rotating member, able to move a fixed volume of samplefrom the first to the second-stage PCR reagent. A one microliterfraction of the pre-amplified sample, mixed with 100 microliters offresh PCR reagent would yield a one-hundred-fold reduction inconcentration. It is understood that this dilution is illustrative only,and that other volumes and dilution levels are possible. This approachcould be accomplished by forcing the first-stage amplification productinto the rigid fitment where it contacts one of the plungers 68 or 69 ofFIG. 1. In such an embodiment, the plunger would be configured to carrya small fraction of the sample into contact with the adjacent dilutionbuffer or second-stage PCR buffer. Similarly a sliding element could beused to carry a small amount of the first-stage amplification productinto contact with the second-stage reaction mix while maintaining a sealbetween the stages, and containing the amplified sample within the rigidfitment 90.

Subsequent to first-stage PCR and dilution, channel 78 transfers thesample to a plurality of low volume blisters 82 for secondary nestedPCR. In one illustrative embodiment, dried primers provided in thesecond-stage blisters are resuspended by the incoming aqueous materialto complete the reaction mixture. Optionally, fluorescent dyes such asLCGreen® Plus (Idaho Technology, Salt Lake City, Utah) used fordetection of double-stranded nucleic acid may be provided in eachblister or may be added to the incomplete PCR master mix provided at theend of the serial dilution, although it is understood that LCGreen® Plusis illustrative only and that other dyes are available, as are known inthe art. In another optional embodiment, dried fluorescently labeledoligonucleotide probes configured for each specific amplicon may beprovided in each respective second-stage blister, along with therespective dried primers. Further, while pouch 10 is designed to containall reactions and manipulations within, to reduce contamination, in somecircumstances it may be desirable to remove the amplification productsfrom each blister 82 to do further analysis. Other means for detectionof the second-stage amplicon, as are known in the art, are within thescope of this invention. Once the sample is transferred to blisters 82,pinch valves 84 and 86 are activated to close off blisters 82. Eachblister 82 now contains all reagents needed for amplification of aparticular target. Illustratively, each blister may contain a uniquepair of primers, or a plurality of blisters 82 may contain the sameprimers to provide a number of replicate amplifications.

It is noted that the embodiments disclosed herein use blisters for thesecond-stage amplification, wherein the blisters are formed of the sameor similar plastic film as the rest of the flexible portion. However, inmany embodiments, the contents of the second-stage blisters are neverremoved from the second-stage blisters, and, therefore, there is no needfor the second-stage reaction to take place in flexible blisters. It isunderstood that the second-stage reaction may take place in a pluralityof rigid, semi-rigid, or flexible chambers that are fluidly connected tothe blisters. The chambers could be sealed as in the present example byplacing pressure on flexible channels that connect the chambers, or maybe sealed in other ways, illustratively by heat sealing or use ofone-way valves. Various embodiments discussed herein include blistersprovided solely for the collection of waste. Since the waste may neverbe removed, waste could be collected in rigid, semi-rigid, or flexiblechambers.

It is within the scope of this invention to do the second-stageamplification with the same primers used in the first-stageamplification (see U.S. Pat. No. 6,605,451). However, it is oftenadvantageous to have primers in second-stage reactions that are internalto the first-stage product such that there is no or minimal overlapbetween the first- and second-stage primer binding sites. Dilution offirst-stage product largely eliminates contribution of the originaltemplate DNA and first-stage reagents to the second-stage reaction.Furthermore, illustratively, second-stage primers with a Tm higher thanthose used in the first-stage may be used to potentiate nestedamplification. Primers may be designed to avoid significant hairpins,hetero/homo-dimers and undesired hybridization. Because of the nestedformat, second-stage primers tolerate deleterious interactions far moreso than primers used to amplify targets from genomic DNA in a singlestep. Optionally, hot-start is Used on second-stage amplification.

If a fluorescent dye is included in second-stage amplification,illustratively as a dsDNA binding dye or as part of a fluorescent probe,as are known in the art, optics provided may be used to monitoramplification of one or more of the samples. Optionally, analysis of theshape of the amplification curve may be provided to call each samplepositive or negative. Illustrative methods of calling the sample arediscussed in U.S. Pat. No. 6,730,501, herein incorporated by reference.Alternatively, methods employing a crossing threshold may be used. Acomputer may be provided externally or within the instrument and may beconfigured to perform the methods and call the sample positive ornegative based upon the presence or absence of second-stageamplification, and may provide quantitative information about thestarting template concentration by comparing characteristic parametersof the amplification curve (such as crossing threshold) to standardcurves, or relative to other amplification curves within the run. It isunderstood, however, that other methods, as are known in the art, may beused to call each sample. Other analyses may be performed on thefluorescent information. One such non-limiting example is the use ofmelting curve analysis to show proper melting characteristics (e.g. Tm,melt profile shape) of the amplicon. The optics provided may beconfigured to capture images of all blisters 82 at once, or individualoptics may be provided for each individual blister. Other configurationsare within the scope of this invention.

FIG. 5 shows an alternative pouch 110. In this embodiment, variousreagents are loaded into pouch 110 via fitment 190. FIG. 5a shows across-section of fitment 190 with one of a plurality of plungers 168. Itis understood that, while FIG. 5a shows a cross-section through entrychannel 115 a, as shown in the embodiment of FIG. 5, there are 12 entrychannels present (entry channel 115 a through 115 l), each of which mayhave its own plunger 168 for use in fitment 190, although in thisparticular configuration, entry channels 115 c, 115 f, and 115 i are notused. It is understood that a configuration having 12 entry channels isillustrative only, and that any number of entry channels and associatedplungers may be used. In the illustrative embodiment, an optional vacuumport 142 of fitment 190 is formed through a first surface 194 of fitment190 to communicate with chamber 192. Optional vacuum port 142 may beprovided for communication with a vacuum or vacuum chamber (not shown)to draw out the air from within pouch 110 to create a vacuum withinchamber 192 and the various blisters and chambers of pouch 110. Plunger168 is then inserted far enough into chamber 192 to seal off vacuum port142. Chamber 192 is illustratively provided under a predetermined amountof vacuum to draw a desired volume of liquid into chamber 192 upon use.Additional information on preparing chamber 192 may be found in U.S.patent application Ser. No. 10/512,255, already incorporated byreference.

Illustrative fitment 190 further includes an injection port 141 formedin the second surface 195 of fitment 190. Illustratively, injection port141 is positioned closer to the plastic film portion of pouch 110 thanvacuum port 142, as shown in FIG. 5a , such that the plunger 168 isinserted far enough to seal off vacuum port 142, while still allowingaccess to chamber 192 via injection port 141. As shown, second surface119 of plastic film portion 117 provides a penetrable seal 139 toprevent communication between chamber 192 and the surrounding atmospherevia injection port 141. However, it is understood that second surface119 optionally may not extend to injection port 141 and various otherseals may be employed. Further, if another location for the seal isdesired, for example on a first surface 194 of fitment 190, injectionport 141 may include a channel to that location on fitment 190. U.S.patent application Ser. No. 10/512,255, already incorporated byreference, shows various configurations where the seal is locatedremotely from the injection port, and the seal is connected to thechamber via a channel. Also, U.S. patent application Ser. No. 10/512,255discloses various configurations where channels connect a single seal tomultiple chambers. Variations in seal location, as well as connection ofa single injection port to multiple chambers, are within the scope ofthis invention. Optionally, seal 139 may be frangible and may be brokenupon insertion of a cannula (not shown), to allow a fluid sample fromwithin the cannula to be drawn into or forced into chamber 192.

The illustrative plunger 168 of the pouch assembly 110 is cylindrical inshape and has a diameter of approximately 5 mm to be press-fit intochamber 192. Plunger 168 includes a first end portion 173 and anopposite second end portion 175. As shown in FIG. 5a , a notch 169 ofplunger 168 is formed in second end portion 175. In use, second endportion 175 is inserted part way into chamber 192, and notch 169 may bealigned with injection port 141 to allow a fluid sample to be drawn intoor injected into chamber 192, even when plunger 168 is inserted farenough that plunger 168 would otherwise be blocking injection port 141.

Illustratively, a fluid is placed in a container (not shown) with asyringe having a cannulated tip that can be inserted into injection port141 to puncture seal 139 therein. In using an air-evacuated pouchassembly 110, when seal 139 is punctured, the fluid is withdrawn fromthe container due to the negative pressure within chamber 192 relativeto ambient air pressure. Fluid then passes through port 141 to fillchamber 192. At this point, the fluid usually does not flow into theplastic film portion 117 of pouch 110. Finally, the plunger 168 isinserted into chamber 192 such that second end portion 175 of plunger168 approaches the bottom 191 of chamber 192, to push a measured amountof the reagent or sample into the plastic film portion 117. As shown,plunger 168 is configured such that upon full insertion, second endportion 175 does not quite meet bottom 191 of chamber 192. The remainingspace is useful in trapping bubbles, thereby reducing the number ofbubbles entering plastic film portion 117. However, in some embodimentsit may be desirable for second end portion 175 to meet bottom 191 uponfull insertion of plunger 168. In the embodiment shown in FIG. 5, entrychannels 115 a, 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and 115l all lead to reaction zones or reservoir blisters. It is understoodthat full insertion of the plunger associated with entry channel 115 awould force a sample into three-lobed blister 122, full insertion of theplunger associated with entry channel 115 b would force a reagent intoreservoir blister 101, full insertion of the plunger associated withentry channel 115 d would force a reagent into reservoir blister 102,full insertion of the plunger associated with entry channel 115 e wouldforce a reagent into reservoir blister 103, full insertion of theplunger associated with entry channel 115 g would force a reagent intoreservoir blister 104, full insertion of the plunger associated withentry channel 115 h would force a reagent into reservoir blister 105,full insertion of the plunger associated with entry channel 115 j wouldforce a reagent into reservoir blister 106, full insertion of theplunger associated with entry channel 115 k would force a reagent intoreservoir blister 107, and full insertion of the plunger associated withentry channel 115 l would force a reagent into reservoir blister 108.

If a plunger design is used including notch 169 as illustrated in theembodiment shown in FIG. 5a , the plunger 168 may be rotated prior tobeing lowered, so as to offset notch 169 and to close off injection port141 from communication with chamber 192, to seal the contents therein.This acts to minimize any potential backflow of fluid through injectionport 141 to the surrounding atmosphere, which is particularly usefulwhen it is desired to delay in full insertion of the plunger. Althoughnotch 169 is shown and described above with respect to plunger 168, itis within the scope of this disclosure to close off injection port 141soon after dispensing the fluid sample into the chamber 192 by othermeans, such as depressing plunger 168 toward the bottom of chamber 192,heat sealing, unidirectional valves, or self-sealing ports, for example.If heat sealing is used as the sealing method, a seal bar could beincluded in the instrument such that all chambers are heat sealed uponinsertion of the pouch into the instrument.

In the illustrative method, the user injects the sample into theinjection port 141 associated with entry channel 115 a, and water intothe various other injection ports. The water rehydrates reagents thathave been previously freeze-dried into chambers 192 associated with eachof entry channels 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and115 l. The water may be injected through one single seal and then bedistributed via a channel to each of the chambers, as shown in FIG. 6below, or the water could be injected into each chamber independently.Alternatively, rather than injecting water to rehydrate dried reagents,wet reagents such as lysis reagents, nucleic acid extraction reagents,and PCR reagents may be injected into the appropriate chambers 192 ofthe fitment 190.

Upon activation of the plunger 168 associated with entry channel 115 a,the sample is forced directly into three-lobed blister 122 via channel114. The user also presses the remaining plungers 168, forcing thecontents out of each of the chambers 192 in fitment 190 and intoreservoir blisters 101 through 108. At this point, pouch 110 is loadedinto an instrument for processing. While instrument 800, shown in FIG.8, is configured for the pouch 210 of FIG. 6, it is understood thatmodification of the configuration of the bladders of instrument 800would render instrument 800 suitable for use with pouches 110 and 510,or with pouches of other configurations.

In one illustrative example, upon depression of the plungers 168,reservoir blister 101 now contains DNA-binding magnetic beads inisopropanol, reservoir blister 102 now contains a first wash solution,reservoir blister 103 now contains a second wash solution, reservoirblister 104 now contains a nucleic acid elution buffer, reservoirblister 105 now contains first-stage PCR reagents, including multiplexedfirst-stage primers, reservoir blister 106 now contains second-stage PCRreagents without primers, reservoir blister 107 now contains negativecontrol PCR reagents without primers and without template, and reservoirblister 108 now contains positive control PCR reagents with template.However, it is understood that these reagents are illustrative only, andthat other reagents may be used, depending upon the desired reactionsand optimization conditions.

Once pouch 110 has been placed into instrument 800 and the sample hasbeen moved to three-lobed blister 122, the sample may be subjected todisruption by agitating the sample with lysing particles such as ZS orceramic beads. The lysing particles may be provided in three-lobedblister 122, or may be injected into three-lobed blister 122 along withthe sample. The three-lobed blister 122 of FIG. 5 is operated in muchthe same way as three-lobed blister 22 of FIG. 1, with the two lowerlobes 124, 126 pressurized together, and pressure is alternated betweenthe upper lobe 128 and the two lower lobes 124, 126. However, asillustrated, lower lobes 124, 126 are much more rounded than lower lobes24, 26, allowing for a smooth flow of beads to channel 130 and allowingfor high-speed collisions, even without the triangular flow separator atnexus 32. As with three-lobed blister 22, three-lobed blister 122 ofFIG. 5 allows for effective lysis or disruption of microorganisms,cells, and viral particles in the sample. It has been found that achannel 130 having a width of about 3-4 mm, and illustratively about 3.5mm, remains relatively clear of beads during lysis and is effective inproviding for high-velocity collisions.

After lysis, nucleic-acid-binding magnetic beads are injected into upperlobe 128 via channel 138 by pressurizing a bladder positioned overreservoir blister 101. The magnetic beads are mixed, illustratively moregently than with during lysis, with the contents of three-lobed blister122, and the solution is incubated, illustratively for about 1 minute,to allow nucleic acids to bind to the beads.

The solution is then pumped into the “figure 8” blister 144 via channel143, where the beads are captured by a retractable magnet housed in theinstrument, which is illustratively pneumatically driven. The beadcapture process begins by pressurizing all lobes 124, 126, and 128 ofthe bead milling apparatus 122. This forces much of the liquid contentsof 122 through channel 143 and into blister 144. A magnet is broughtinto contact with the lower portion 144 b of blister 144 and the sampleis incubated for several seconds to allow the magnet to capture thebeads from the solution, then the bladders adjacent to blister 122 aredepressurized, the bladders adjacent blister portions 144 a and 144 bare pressurized, and the liquid is forced back into blister 122. Sincenot all of the beads are captured in a single pass, this process may berepeated up to 10 times to capture substantially all of the beads inblister 144. Then the liquid is forced out of blister 144, leavingbehind only the magnetic beads and the captured nucleic acids, and washreagents are introduced into blister 144 in two successive washes (fromreservoir blisters 102 and 103 via channels 145 and 147, respectively).In each wash, the bladder positioned over the reservoir blistercontaining the wash reagent is pressurized, forcing the contents intoblister 144. The magnet is withdrawn and the pellet containing themagnetic beads is disrupted by alternatively pressurizing each of twobladders covering each lobe 144 a and 144 b of blister 144. When theupper lobe 144 a is compressed, the liquid contents are forced into thelower lobe 144 b, and when the lower lobe 144 b is compressed, thecontents are forced into the upper lobe 144 a. By agitating the solutionin blister 144 between upper lobe 144 a and lower lobe 144 b, themagnetic beads are effectively washed of impurities. A balance ismaintained between inadequate agitation, leaving the pellet of beadsundisturbed, and excessive agitation, potentially washing the nucleicacids from the surface of the beads and losing them with the washreagents. After each wash cycle, the magnetic beads are captured via themagnet in blister 144 and the wash reagents are illustratively forcedinto three-lobed blister 122, which now serves as a waste receptacle.However, it is understood that the used reservoir blisters may alsoserve as waste receptacles, or other blisters may be providedspecifically as waste receptacles.

Nucleic acid elution buffer from reservoir blister 104 is then injectedvia channel 149 into blister 144, the sample is once again agitated, andthe magnetic beads are recaptured by employment of the magnet. The fluidmixture in blister 144 now contains nucleic acids from the originalsample. Pressure on blister 144 moves the nucleic acid sample to thefirst stage PCR blister 161 via channel 152, where the sample is mixedwith first-stage PCR master mix containing multiple primer sets, the PCRmaster mix provided from reservoir blister 105 via channel 162. Ifdesired, the sample and/or the first-stage PCR master mix may be heatedprior to mixing, to provide advantages of hot start. Optionally,components for reverse transcription of RNA targets may be providedprior to first-stage PCR. Alternatively, an RT enzyme, illustratively athermostable RT enzyme may be provided in the first-stage PCR master mixto allow for contemporaneous reverse transcription of RNA targets. It isunderstood that an RT enzyme may be present in the first-stage PCRmixture in any of the embodiments disclosed herein. As will be seenbelow, pouch 110 of FIG. 5 is configured for up to 10 primer sets, butit is understood that the configuration may be altered and any number ofprimer sets may be used. A bladder positioned over blister 161 ispressurized at low pressure, to force the contents of blister 161 intointimate contact with a heating/cooling element, illustratively aPeltier element, on the other side of blister 161. The pressure onblister 161 should be sufficient to assure good contact with theheating/cooling element, but should be gentle enough such that fluid isnot forced from blister 161. The heating/cooling element is temperaturecycled, illustratively between about 60° C. to about 95° C.Illustratively, temperature cycling is performed for about 15-20 cycles,resulting in amplification of one or more nucleic acid targets present.Also illustratively, temperature cycling ceases prior to plateau phase,and may cease in log phase or even prior to log phase. In one example,it may be desirable merely to enrich the sample with the desiredamplicons, without reaching minimal levels of detection. See U.S. Pat.No. 6,605,451, herein incorporated by reference.

The amplified sample is optionally then diluted by forcing most thesample back into blister 144 via channel 152, leaving only a smallamount (illustratively about 1 to 5%) of the amplified sample in blister161, and second-stage PCR master mix is provided from reservoir blister106 via channel 163. The sample is thoroughly mixed illustratively bymoving it back and forth between blisters 106 and 161 via channel 163.If desired, the reaction mixture may be heated to above extensiontemperature, illustratively at least 60° C., prior to second-stageamplification. The sample is then forced through channel 165 into anarray of low volume blisters 182 in the center of second-stageamplification zone 180. Each of the ten illustrative low volume blisters182 may contain a different primer pair, either essentially the same asone of the primer pairs in the first-stage amplification, or “nested”within the first-stage primer pair to amplify a shortened amplicon. Theprimers, now hydrated by the sample, complete the amplification mixture.Positive and negative control samples are also introduced bypressurizing the contents of reservoir blisters 107 and 108,respectively, forcing PCR master mix either without target DNA fromreservoir blister 107 via channel 166, or with control DNA fromreservoir blister 108, via channel 167. As illustrated, there are fiveeach of positive control blisters 183 and negative control blisters 181,which may be multiplexed 2-fold to provide the necessary controls forten different second-stage amplification reactions. It is understoodthat this configuration is illustrative only and that any number ofsecond-stage blisters may be provided.

Illustratively, the PCR master mix used for second-stage amplificationlacks the primers, but is otherwise complete. However, an “incomplete”PCR master mix may lack other PCR components as well. In one example,the second-stage PCR master mix is water or buffer only, which is thenmixed with the optionally diluted first-stage PCR amplification product.This mixture is moved to the small-volume PCR reaction blisters, whereall of the remaining components have been previously provided. Ifdesired, all of the remaining components may be mixed together andspotted as a single mixture into the small-volume PCR reaction blisters.Alternatively, as illustrated in FIG. 5b , each of the components may bespotted onto a separate region of the small-volume PCR reaction blister182. As shown in FIG. 5b , four regions are present, illustratively withdNTPs spotted at region 182 a, primers spotted at 182 b, polymerasespotted at 182 c, and a magnesium compound spotted at 182 d. By spottingthe components separately and heating the sample mixture prior torehydrating the components, nonspecific reactions can be minimized. Itis understood that any combination of components can be spotted thisway, and that this method of spotting components into one or moreregions of the blisters may be used with any embodiment of the presentinvention.

The channels 165, 166, and 167 leading to the small-volume PCR reactionblisters 181, 182, and 183 are sealed, and a pneumatic bladder gentlypresses the array against a heating/cooling element, illustratively aPeltier element, for thermal cycling. The cycling parameters may beindependently set for second-stage thermal cycling. Illustratively, thereactions are monitored by focusing an excitation source, illustrativelya blue light (450-490 nm), onto the array, and imaging the resultantfluorescent emissions, illustratively fluorescent emissions above 510nm.

In the above example, pinch valves are not discussed. However, it isunderstood that when it is desired to contain a sample in one of theblisters, pneumatic actuators positioned over channels leading to andfrom the particular blister are pressurized, creating pinch valves andclosing off the channels. Conversely, when it is desired to move asample from one of the blisters, the appropriate pneumatic actuator isdepressurized, allowing the sample flow through the channel.

The pouch described above in FIG. 5 includes reagent reservoir blisters101 through 108, in which the user injected reagents from the fitment190 into the reagent reservoir blisters 101 through 108 in the plasticfilm portion 117 of the pouch 110, illustratively prior to insertion ofpouch 110 into the instrument. While there are advantages to the use ofthe reagent reservoir blisters of FIG. 5, including having the abilityto maintain the contents of the various blisters at differenttemperatures, there are some disadvantages as well. Because the operatoris responsible for moving the reagents from the fitment 190 to thereservoir blisters 101 through 108, and because this is often doneoutside of the machine and thus without activated pinch valves, reagentscould occasionally leak from the reservoir blisters to the workingblisters. The reagents in reservoir blisters are exposed duringpreparation and loading. If they are pressed, squeezed, or even lightlybumped, the reagents may leak through available channels. If the loss ofreagents is substantial, the reaction may fail completely. Furthermore,during operation there may be some variability in the amount of reagentforced from the reservoir blisters 101 through 108, leading toinconsistent results. Automation of introduction of the reagents tofitment 190 and movement of the reagents from fitment 190 to reagentreservoir blisters 101 through 108 would solve many of these problems,and is within the scope of this invention.

The pouch 210 of FIG. 6 addresses many of these issues in a differentway, by using a direct-injection approach wherein the instrument itselfmoves the plungers 268, illustratively via pneumatic pistons, and forcesthe reagents into the various working blisters as the reagents areneeded. Rather than storing the reagents in reservoir blisters 101through 108 of FIG. 5, in the embodiment of FIG. 6 the reagents areintroduced into various chambers 292 of fitment 290 and are maintainedthere until needed. Pneumatic operation of piston 268 at the appropriatetime introduces a measured amount of the reagent to the appropriatereaction blister. In addition to addressing many of the above-mentionedissues, pouch 210 also has a much more compact shape, allowing for asmaller instrument design, and pouch 210 has shorter channels,permitting better fluid flow and minimizing reagent loss in channels.

In one illustrative embodiment of FIG. 6, a 300 μl mixture comprisingthe sample to be tested (100 μl) and lysis buffer (200 μl) is injectedinto injection port 241 a. Water is also injected into the fitment 290via seal 239 b, hydrating up to eleven different reagents, each of whichwere previously provided in dry form in chambers 292 b through 292 l viachannel 293 (shown in shadow). These reagents illustratively may includefreeze-dried PCR reagents, DNA extraction reagents, wash solutions,immunoassay reagents, or other chemical entities. For the example ofFIG. 6, the reagents are for nucleic acid extraction, first-stagemultiplex PCR, dilution of the multiplex reaction, and preparation ofsecond-stage PCR reagents, and control reactions. In the embodimentshown in FIG. 6, all that need be injected is the sample in port 241 aand water in port 241 b.

As shown in FIG. 6, water injected via seal 293 b is distributed tovarious chambers via channel 293. In this embodiment, only the sampleand water need be injected into pouch 210. It is understood, however,that water could be injected into each chamber 292 independently.Further, it is understood that, rather than providing dried reagents inthe various chambers 292 and hydrating upon injection of the water,specific wet reagents could be injected into each chamber, as desired.Additionally, it is understood that one or more of chambers 292 could beprovided with water only, and the necessary reagents may be provideddried in the appropriate reaction blisters. Various combinations of theabove, as dictated by the needs of the particular reaction, are withinthe scope of this invention.

As seen in FIG. 6, optional protrusions 213 are provided on bottomsurface 297 of fitment 290. As shown, protrusions 213 are located withintheir respective entry channels 215. However, other configurations arepossible. Protrusions 213 assist in opening entry channel 215 andprevent bottom surface 297 from engaging another flat surface in such away to pinch off entry channels 215 when plungers 268 are depressed,which helps prevent back-flow upon activation of the plungers 268. Suchprotrusions may be used on any of the various pouches according to thepresent invention.

In embodiments wherein water is injected into the pouch to hydratemultiple dry reagents in multiple chambers in the fitment, a means ofclosing the channel between the injection port and the many chambers isdesired. If the channel is not closed, activation of each plunger mayforce some of the contents of its respective chamber back out into thechannel, potentially contaminating neighboring chambers and altering thevolumes contained in and delivered from the chamber. Several ways ofclosing this channel have been used, including rotating a notchedplunger 268 as discussed above, and heat-sealing the plastic film acrossthe channel thereby closing the channel permanently, and applyingpressure to the channel as a pinch valve. Other closures may also beused, such as valves built into the fitment, illustratively one-wayvalves.

After the fluids are loaded into chambers 292 and pouch 210 is loadedinto the instrument, plunger 268 a is depressed illustratively viaactivation of a pneumatic piston, forcing the balance of the sample intothree-lobed blister 220 via channel 214. As with the embodiments shownin FIGS. 1 and 5, the lobes 224, 226, and 228 of three-lobed blister 220are sequentially compressed via action bladders 824, 826, and 828 ofbladder assembly 810, shown in FIGS. 7-9, forcing the liquid through thenarrow nexus 232 between the lobes, and driving high velocitycollisions, shearing the sample and liberating nucleic acids,illustratively including nucleic acids from hard-to-open spores,bacteria, and fungi. Cell lysis continues for an appropriate length oftime, illustratively 0.5 to 10 minutes.

Once the cells have been adequately lysed, plunger 268 b is activatedand nucleic acid binding magnetic beads stored in chamber 292 b areinjected via channel 236 into upper lobe 228 of three-lobed blister 220.The sample is mixed with the magnetic beads and the mixture is allowedto incubate for an appropriate length of time, illustrativelyapproximately 10 seconds to 10 minutes.

The mixture of sample and beads are forced through channel 238 intoblister 244 via action of bladder 826, then through channel 243 and intoblister 246 via action of bladder 844, where a retractable magnet 850located in instrument 800 adjacent blister 245, shown in FIG. 8,captures the magnetic beads from the solution, forming a pellet againstthe interior surface of blister 246. A pneumatic bladder 846, positionedover blister 246 then forces the liquid out of blister 246 and backthrough blister 244 and into blister 222, which is now used as a wastereceptacle. However, as discussed above with respect to FIG. 5, otherwaste receptacles are within the scope of this invention. One ofplungers 268 c, 268 d, and 268 e may be activated to provide a washsolution to blister 244 via channel 245, and then to blister 246 viachannel 243. Optionally, the magnet 850 is retracted and the magneticbeads are washed by moving the beads back and forth from blisters 244and 246 via channel 243, by alternatively pressurizing bladders 844 and846. Once the magnetic beads are washed, the magnetic beads arerecaptured in blister 246 by activation of magnet 850, and the washsolution is then moved to blister 222. This process may be repeated asnecessary to wash the lysis buffer and sample debris from the nucleicacid-binding magnetic beads. Illustratively, three washes are done, oneeach using wash reagents in chambers 292 c, 292 d, and 292 e. However,it is understood that more or fewer washes are within the scope of thisinvention. If more washes are desired, more chambers 292 may beprovided. Alternatively, each chamber 292 may hold a larger volume offluid and activation of the plungers may force only a fraction of thevolume from the chamber upon each activation.

After washing, elution buffer stored in chamber 292 f is moved viachannel 247 to blister 248, and the magnet is retracted. The solution iscycled between blisters 246 and 248 via channel 252, breaking up thepellet of magnetic beads in blister 246 and allowing the capturednucleic acids to dissociate from the beads and come into solution. Themagnet 850 is once again activated, capturing the magnetic beads inblister 246, and the eluted nucleic acid solution is forced into blister248.

Plunger 268 h is depressed and first-stage PCR master mix from chamber292 h is mixed with the nucleic acid sample in blister 248. Optionally,the mixture is mixed by alternative activation of bladders 848 and 864,forcing the mixture between 248 and 264 via channel 253. After severalcycles of mixing, the solution is contained in blister 264, wherefirst-stage multiplex PCR is performed. If desired, prior to mixing, thesample may be retained in blister 246 while the first-stage PCR mastermix is pre-heated, illustratively by moving the first-stage PCR mastermix into blister 264 or by providing a heater adjacent blister 248. Asdiscussed above, this pre-heating may provide the benefits of hot startPCR. The instrument 800 illustrated in FIG. 8 features Peltier-basedthermal cyclers 886 and 888 which heat and cool the sample. However, itis understood that other heater/cooler devices may be used, as discussedabove. Optionally, mixing between blisters 248 and 264 may continueduring temperature cycling, with thermal cycler 886 positioned to heatand cool both blisters 248 and 264. It has been found that such mixingimproves the first-stage PCR reaction in some embodiments. Also, thermalcycling can be accomplished by varying the temperatures in two or moredifferent blisters, allowing minimal energy expenditure and maximizingthermal cycling speed. For example the temperature can be maintained at95° C. in blister 248, and 65° C. blister 264, and moving the samplebetween these blisters effectively transfers heat into and out of thesample, allowing rapid and accurate thermal cycling. Temperature cyclingis illustratively performed for 15-20 cycles, although other levels ofamplification may be desirable, depending on the application, asdiscussed above. As will be seen below, the second-stage amplificationzone 280 is configured to detect amplification in 18 second-stagereactions. Accordingly, 18 different primer-pairs may be included in thePCR reaction in blister 264.

In an alternative hot start method, pouch 210 is manufactured with theprimers provided in one of the blisters, illustratively blister 264. Inone embodiment, the primers are freeze dried separately and thenintroduced during manufacture into blister 264 as a friable pellet.Prior to first-stage PCR, illustratively the sample is eluted fromblister 246 and pushed to blister 264 to rehydrate the primer pellet.Peltier 886, which is positioned adjacent blisters 248 and 264 is heatedto 48° C., and PCR master mix is pushed to blister 248. After a hold,illustratively for 10 seconds, during which the two blisters reach 48°C., mixing between blisters 248 and 264 begins. Thus, the enzymes anddNTPs remain in blister 248 and most of the sample and the primersremain in blister 264 until the components separately have reached 48°C. It is understood, however, that the choice of 48° C. was made for usewith concurrent first-stage amplification and RT using AMY, which isactive up to 50° C. If RT is not needed or a more thermostable RT enzymeis used, then one or both of the two blisters 248 and 264 may be heatedup to 58° C., or even higher, depending on the primer meltingtemperature or other factors in a particular first-stage amplificationprotocol. It is understood that this hot start method may be used withany embodiment of the present invention.

In an alternative embodiment, to reduce the complexity of thefirst-stage PCR reaction, blister 248 may be divided into two or moreblisters. It is believed that the number of nonspecific products of amultiplex reaction goes up as the square (or possibly higher power) ofthe number of primers in the mixture, while the loss of sensitivity ofan assay is a linear function of the amount of input sample. Thus, forexample, splitting the first stage PCR into two reactions, each of halfthe volume of the single reaction of this embodiment, would reducesensitivity by two-fold but the quantity and complexity of thenonspecific reactions would be ¼ as much. If blister 248 is divided intoor more blisters, blister 264 may be divided into a number of blistersequal to the number of blisters 248. Each respective blister 248 wouldbe connected to its respective blister 264 via a respective channel 253.Each blister 264 would be provided with a pellet comprising a subset ofall primers. Sample from blister 246 would be divided across eachblister 248, each blister 248 would be sealed from all others, andthermal cycling would proceed with each pair of blisters 248 and 264, asdescribed above. After thermal cycling, the sample would be recombinedinto blister 266 or individually sent to separate sets of second-stageblisters.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted as discussed above with respect to theembodiment of FIG. 5, by forcing most of the sample back into blister248, leaving only a small amount, and adding second-stage PCR master mixfrom chamber 292 i. Alternatively, a dilution buffer from 292 i may bemoved to blister 266 via channel 249 and then mixed with the amplifiedsample in blister 264 by moving the fluids back and forth betweenblisters 264 and 266. After mixing, a portion of the diluted sampleremaining in blister 264 is forced away to three-lobed blister 222, nowthe waste receptacle. If desired, dilution may be repeated severaltimes, using dilution buffer from chambers 292 j and 292 k, and thenadding second-stage PCR master mix from chamber 292 g to some or all ofthe diluted amplified sample. It is understood that the level ofdilution may be adjusted by altering the number of dilution steps or byaltering the percentage of the sample discarded prior to mixing with thedilution buffer or second-stage PCR master mix. If desired, this mixtureof the sample and second-stage PCR master mix may be pre-heated inblister 264 prior to movement to second-stage blisters 282 forsecond-stage amplification. As discussed above, such preheating mayobviate the need for a hot-start component (antibody, chemical, orotherwise) in the second-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 18 second-stage blisters 282 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may then bepre-loaded in the second-stage blisters 282 as well. As discussed abovewith the prior examples, each primer pair may be identical to afirst-stage PCR primer pair or may be nested within the first-stageprimer pair. Movement of the sample from blister 264 to the second-stageblisters completes the PCR reaction mixture. Control samples fromchamber 292 l are also moved to control blisters 283 via channel 267.The control samples may be positive or negative controls, as desired.Illustratively, each pouch would contain control reactions that validatethe operation of each step in the process and demonstrate that positiveresults are not the result of self-contamination with previouslyamplified nucleic acids. However, this is not practical in manyprotocols, particularly for a highly multiplexed reaction. Oneillustrative way of providing suitable controls involves spiking sampleswith a species such as baker's yeast. The nucleic acids are extractedfrom the yeast, alongside other nucleic acids. First- and second-stagePCR reactions amplify DNA and/or RNA targets from the yeast genome.Illustratively, an mRNA sequence derived from a spliced pre-mRNA can beused to generate an RNA-specific target sequence by arranging the primersequences to span an intron. A quantitative analysis of the yeast copynumber against reference standards allows substantial validation thateach component of the system is working. Negative control reactions foreach of the many second-stage assays are more problematic. It may bedesirable to run control reactions either in parallel or in a separaterun.

Activation of bladder 882 of bladder assembly 810 seals the samples intotheir respective second-stage blisters 282, 283, and activation ofbladder 880 provides gentle pressure on second-stage blisters 282, 283,to force second-stage blisters 282, 283 into contact with aheater/cooler device. A window 847 positioned over the second-stageamplification zone 280 allows fluorescence monitoring of the arrayduring PCR and during a DNA melting-curve analysis of the reactionproducts.

It is noted that the pouch 210 of FIG. 6 has several unsealed areas,such as unsealed area 255 and unsealed area 256. These unsealed areasform blisters that are not involved in any of the reactions in thisillustrative embodiment. Rather, these unsealed areas are provided inspace between the working blisters and channels. In some manufacturingprocesses, as compared to pouches that are sealed in all unused space,it has been found that fewer leaks sometimes result when unsealed areassuch as 255 and 256 are provided, presumably by reducing problematicwrinkles in the film material. Such unsealed areas optionally may beprovided on any pouch embodiment.

FIG. 8 shows an illustrative apparatus 800 that could be used with pouch210. Instrument 800 includes a support member 802 that could form a wallof a casing or be mounted within a casing. Instrument 800 also includesa second support member 804 that is optionally movable with respect tosupport member 802, to allow insertion and withdrawal of pouch 210.Movable support member 804 may be mounted on a track or may be movedrelative to support member 802 in any of a variety of ways.Illustratively, a lid 805 fits over pouch 210 once pouch 210 has beeninserted into instrument 800. In another embodiment, both supportmembers 802 and 804 may be fixed, with pouch 210 held into place byother mechanical means or by pneumatic pressure.

Illustratively, the bladder assembly 810 and pneumatic valve assembly808 are mounted on movable member 802, while the heaters 886 and 888 aremounted on support member 802. However, it is understood that thisarrangement is illustrative only and that other arrangements arepossible. As bladder assembly 810 and pneumatic valve assembly 808 aremounted on movable support member 804, these pneumatic actuators may bemoved toward pouch 210, such that the pneumatic actuators are placed incontact with pouch 210. When pouch 210 is inserted into instrument 800and movable support member 804 is moved toward support member 802, thevarious blisters of pouch 210 are in a position adjacent to the variouspneumatic bladders of bladder assembly 810 and the various pneumaticpistons of pneumatic valve assembly 808, such that activation of thepneumatic actuators may force liquid from one or more of the blisters ofpouch 210 or may form pinch valves with one or more channels of pouch210. The relationship between the blisters and channels of pouch 210 andthe pneumatic actuators of bladder assembly 810 and pneumatic valveassembly 808 are discussed in more detail below with respect to FIGS. 9and 10.

Each pneumatic actuator has one or more pneumatic fittings. For example,bladder 824 of bladder assembly 810 has pneumatic fitting 824 a andpneumatic piston 843 has its associated pneumatic fitting 843 a. In theillustrative embodiment, each of the pneumatic fittings of bladderassembly 810 extends through a passageway 816 in movable support member804, where a hose 878 connects each pneumatic fitting to compressed airsource 895 via valves 899. In the illustrative embodiment, thepassageways 816 not only provide access to compressed air source 895,but the passageways also aid in aligning the various components ofbladder assembly 810, so that the bladders align properly with theblisters of pouch 210.

Similarly, pneumatic valve assembly 808 is also mounted on movablesupport member 804, although it is understood that other configurationsare possible. In the illustrative embodiment, pins 858 on pneumaticvalve assembly 808 mount in mounting openings 859 on movable supportmember 804, and pneumatic pistons 843, 852, 853, and 862 extend throughpassageways 816 in movable support member 804, to contact pouch 210. Asillustrated, bladder assembly is mounted on a first side 811 of movablesupport member 804 while pneumatic valve assembly 808 is mounted on asecond side 812 of movable support member 804. However, becausepneumatic pistons 843, 852, 853, and 862 extend through passageways 816,the pneumatic pistons of pneumatic valve assembly 808 and the pneumaticbladders of bladder assembly 810 work together to provide the necessarypneumatic actuators for pouch 210.

As discussed above, each of the pneumatic actuators of bladder assembly810 and pneumatic valve assembly 808 has an associated pneumaticfitting. While only several hoses 878 are shown in FIG. 8, it isunderstood that each pneumatic fitting is connected via a hose 878 tothe compressed gas source 895. Compressed gas source 895 may be acompressor, or, alternatively, compressed gas source 895 may be acompressed gas cylinder, such as a carbon dioxide cylinder. Compressedgas cylinders are particularly useful if portability is desired. Othersources of compressed gas are within the scope of this invention.

Several other components of instrument 810 are also connected tocompressed gas source 895. Magnet 850, which is mounted on a first side813 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 246 of pouch 210. However, depending on the material ofsupport member 802, it is understood that recess 851 need not extend allthe way through support member 802, as long as when magnet 850 isdeployed, magnet 850 is close enough to provide a sufficient magneticfield at blister 246, and when magnet 850 is retracted, magnet 850 doesnot significantly affect any magnetic beads present in blister 246.While reference is made to retracting magnet 850, it is understood thatan electromagnet may be used and the electromagnet may be activated andinactivated by controlling flow of electricity through theelectromagnet. Thus, while this specification discusses withdrawing orretracting the magnet, it is understood that these terms are broadenough to incorporate other ways of withdrawing the magnetic field. Itis understood that the pneumatic connections may be pneumatic hoses orpneumatic air manifolds, thus reducing the number of hoses or valvesrequired.

The various pneumatic pistons 868 of pneumatic piston array 869, whichis mounted on support 802, are also connected to compressed gas source895 via hoses 878. While only two hoses 878 are shown connectingpneumatic pistons 868 to compressed gas source 895, it is understoodthat each of the pneumatic pistons 868 are connected to compressed gassource 895. Twelve pneumatic pistons 868 are shown. When the pouch 210is inserted into instrument 800, the twelve pneumatic pistons 868 arepositioned to activate their respective twelve plungers 268 of pouch210. When lid 805 is closed over pouch 210, a lip 806 on lid 805provides a support for fitment 290, so that as the pneumatic pistons 868are activated, lid 805 holds fitment 290 in place. It is understood thatother supports for fitment 290 are within the scope of this invention.

A pair of heating/cooling devices, illustratively Peltier heaters, aremounted on a second side 814 of support 802. First-stage heater 886 ispositioned to heat and cool the contents of blister 264 for first-stagePCR. Second-stage heater 888 is positioned to heat and cool the contentsof second-stage blisters 282 and 283 of pouch 210, for second-stage PCR.It is understood, however, that these heaters could also be used forother heating purposes, and that other heaters may be included, asappropriate for the particular application.

If desired, a feedback mechanism (not shown) may be included ininstrument 800 for providing feedback regarding whether the sample hasactually been forced into a particular blister. Illustrative feedbackmechanisms include temperature or pressure sensors or optical detectors,particularly if a fluorescent or colored dye is included. Such feedbackmechanisms illustratively may be mounted on either of support members802 or 804. For example, a pressure sensor may be mounted on support 802adjacent the location of blister 264. When the sample is supposedlymoved to blister 264, if the pressure sensor is depressed, then sampleprocessing is allowed to continue. However, if the pressure sensor isnot depressed, then sample processing may be stopped, or an errormessage may be displayed on screen 892. Any combination or all of theblisters may have feedback mechanisms to provide feedback regardingproper movement of the sample through the pouch.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 8, optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. A window 847 through movablesupport 804 provides optical array 890 with access to second-stageamplification zone 280 of pouch 210. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage blister282, 823 in pouch 210. Alternatively, camera 896 may take images thatcontain all of the second-stage blisters 282, 283, and the image may bedivided into separate fields corresponding to each of the second-stageblisters 282, 283. Depending on the configuration, optical array 890 maybe stationary, or optical array 890 may be placed on movers attached toone or more motors and moved to obtain signals from each individualsecond-stage blister 282, 283. It is understood that other arrangementsare possible.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Computer894 also controls heaters 886 and 888, and optical array 890. Each ofthese components is connected electrically, illustratively via cables891, although other physical or wireless connections are within thescope of this invention. It is understood that computer 894 may behoused within instrument 890 or may be external to instrument 890.Further, computer 894 may include built-in circuit boards that controlsome or all of the components, and may also include an externalcomputer, such as a desktop or laptop PC, to receive and display datafrom the optical array. An interface 893, illustratively a keyboardinterface, may be provided including keys for inputting information andvariables such as temperatures, cycle times, etc. Illustratively, adisplay 892 is also provided. Display 892 may be an LED, LCD, or othersuch display, for example.

FIG. 9 shows the relationship between bladder assembly 810 and pouch 210during operation of instrument 800. Bladder assembly comprisessub-assemblies 815, 817, 818, 819, and 822. Because bladder 809 ofbladder sub-assembly 815 is large, bladder sub-assembly 815illustratively has two pneumatic fittings 815 a and 815 b. Bladder 809is used to close off chambers 292 (as shown in FIG. 6) from the plasticfilm portion 217 of pouch 210. When one of the plungers 268 isdepressed, one or both of pneumatic fittings 815 a and 815 b permitbladder 809 to deflate. After the fluid from one of the chambers 292passes through, bladder 809 is re-pressurized, sealing off channels 214,236, 245, 247, and 249. While illustrative bladder sub-assembly 815 hasonly one bladder 809, it is understood that other configurations arepossible, illustratively where each of channels 214, 236, 245, 247, and249 has its own associated bladder or pneumatic piston. Bladdersub-assembly 822 illustratively comprises three bladders 824, 826, and828. As discussed above, bladders 824, 824, and 828 drive thethree-lobed blister 222 for cell lysis. As illustrated, bladders 824,826, and 828 are slightly larger than their corresponding blisters 224,226, 228. It has been found that, upon inflation, the surface of thebladders can become somewhat dome-shaped, and using slightly oversizedbladders allows for good contact over the entire surface of thecorresponding blister, enabling more uniform pressure and betterevacuation of the blister. However, in some circumstances, completeevacuation of individual blisters may or may not be desired, and largeror smaller-sized bladders may be used to control the blister volumeevacuated. Bladder sub-assembly 817 has four bladders. Bladder 836functions as a pinch-valve for channel 236, while bladders 844, 848, and866 are configured to provide pressure on blisters 244, 248, and 266,respectively. Bladder sub-assembly 818 has two bladders 846 and 864,which are configured to provide pressure on blisters 246 and 264,respectively. Finally, bladder sub-assembly 819 controls second-stageamplification zone 280. Bladder 865 acts as a pinch valve for channels265 and 267, while bladder 882 provides gentle pressure to second-stageblisters 282 and 283, to force second-stage blisters into close contactwith heater 888. While bladder assembly 810 is provided with fivesub-assemblies, it is understood that this configuration is illustrativeonly and that any number of sub-assemblies could be used or that bladderassembly 810 could be provided as a single integral assembly.

FIG. 10 similarly shows the relationship between pneumatic valveassembly 808 and pouch 210 during operation of instrument 800. Ratherthan bladders, pneumatic valve assembly 808 has four pneumatic pistons842, 852, 853, and 862. These pneumatic pistons 842, 852, 853, and 862,each driven by compressed air, provide directed pressure on channels242, 252, 253, and 262. Because the pistons are fairly narrow indiameter, they can fit between bladder sub-assembly 817 and bladdersub-assembly 818 to provide pinch valves for channels 242, 252, 253, and262, allowing channels 242, 252, 253, and 262 to be fairly short.However, if desired, pneumatic pistons 842, 852, 853, and 862 could bereplaced by bladders, which may be included in bladder assembly 810,obviating the need for pneumatic valve assembly 808. It is understoodthat any combination of bladders and pneumatic pistons are within thescope of this invention. It is also understood that other methods ofproviding pressure on the channels and blisters of pouch 210, as areknown in the art, are within the scope of this invention.

FIG. 15 shows a pouch 510 that is similar to pouch 210 of FIG. 6.Fitment 590, with entry channels 515 a through 515 l, is similar tofitment 290, with entry channels 215 a through 215 l. Blisters 544, 546,548, 564, and 566, with their respective channels 538, 543, 552, 553,562, and 565 are similar to blisters 244, 246, 248, 264, and 266, withtheir respective channels 238, 243, 252, 253, 262, and 265 of pouch 210.The channels 245, 247, and 249 of pouch 210 have been somewhatreconfigured as channels 545 a-c, 547 a-b, and 548 a-c on pouch 510; therespective channels of 510 are somewhat shorter than their counterpartchannels on pouch 210. However, it is understood that channelconfigurations are illustrative only, and that various channelconfigurations are within the scope of this invention.

There are two main differences between pouch 510 of FIG. 15 and pouch210 of FIG. 6. First, three-lobed blister 222 has been replaced by lysisblister 522. Lysis blister 522 is configured for vortexing via impactionusing rotating blades or paddles 21 attached to electric motor 19, asshown in FIG. 2b . Since this method of lysing does not rely onalternating pressure of pneumatic pistons, only a single-lobed blisteris shown. Because lysis blister 522 has only a single lobe, bothchannels 514 and 536 lead to the single lobe of lysis blister 522. It isunderstood that lysis blister 522 may be used in any of the pouchembodiments described herein. It is further understood that lysisassembly 810 illustratively may be modified to replace bladders 824,826, and 828 of bladder sub-assembly 822 with a single bladderconfigured for blister 522. Conversely, a three-lobed blister, asdescribed in various other embodiments above, may be used in pouch 510.Lysis blister 522 may be provided with an optional reinforcing patch523, illustratively attached using adhesive or lamination to theexterior surface of lysis blister 522. Reinforcing patch 523 aids inminimizing tearing of pouch 510 due to repeated contact by paddles 21.FIG. 15a shows an electric motor, illustratively a Mabuchi RC-280SA-2865DC Motor (Chiba, Japan), mounted on second support member 804. In oneillustrative embodiment, the motor is turned at 5,000 to 25,000 rpm,more illustratively 10,000 to 20,000 rpm, and still more illustrativelyapproximately 15,000 to 18,000 rpm. For the Mabuchi motor, it has beenfound that 7.2V provides sufficient rpm for lysis. It is understood,however, that the actual speed may be somewhat slower when the blades 21are impacting pouch 510. Other voltages and speeds may be used for lysisdepending on the motor and paddles used. Optionally, controlled smallvolumes of air may be provided into the bladder adjacent lysis blister522. It has been found that in some embodiments, partially filling theadjacent bladder with one or more small volumes of air aids inpositioning and supporting lysis blister during the lysis process.Alternatively, other structure, illustratively a rigid or compliantgasket or other retaining structure around lysis blister 522, can beused to restrain pouch 510 during lysis.

The second main difference between pouch 510 of FIG. 15 and pouch 210 ofFIG. 6 is that the blisters 281, 282, and 283 of second-stageamplification zone 280 have been replaced by high density array 581 insecond-stage amplification zone 580. High density array 581 comprises aplurality of second-stage wells 582, illustratively 50 or more wells,and even more illustratively 120 or more wells. Embodiments with moresecond-stage wells 582, illustratively about 200, about 400, or evenabout 500 or more are within the scope of this invention. Otherconfigurations are within the scope of this invention as well.Additional second-stage wells 582 may be added by making wells 582smaller, by making high density array 581 larger, or a combinationthereof. For second-stage PCR, each of the wells may contain a pair ofprimers. It is understood that one or more wells may be used forpositive or negative controls.

Cross-contamination between wells as the wells are filled with thediluted first-stage amplification product in blister 566 can be a majorproblem. Cross-contamination was controlled in pouch 210 by filling eachsecond-stage blister through a separate branch of channel 265 and thensealing with bladder 882, illustrated in FIG. 9. With high density array581, wherein fluid may fill some or all of blister 584,cross-contamination between wells must also be controlled. In oneembodiment, the second-stage PCR primers may be bound covalently ornon-covalently to the inside surface of each well, thus functioning muchlike a PCR chip. However, in many embodiments it is desirable to controlcross-contamination between wells without tethering the PCR primers tothe wells. Controlling cross-contamination between wells can bedifficult in an embodiment wherein the fluid from blister 566 is movedto wells 582 by flowing across a first surface 581 a of high densityarray 581.

There are several desirable features for successful loading of thesecond-stage amplification zone 580. First, it is desirable that theincoming fluid from blister 566 fill substantially all of the wells 582to substantially the same level. An unfilled well would produce a falsenegative signal. Second, it is desirable that the process of filling thewells 582 should not cause the primers in the well to leak out. Loss ofprimers from one well can limit the efficiency of the PCR reaction inthat well and can contaminate neighboring wells. Third, after the wells582 have been filled and PCR started, it is desirable that the wells becompletely sealed from each other. Amplicon leakage out of one well andinto another well can lower signal in the first well and raise signal inthe second well, potentially leading to a false negative in the firstwell and a false positive in the second well. Further, for certain kindsof controls, it is important that amplicon generated in one well notenter another well where it can be further amplified.

Solutions to this problem include use of a barrier layer. In oneexample, the barrier layer is a physical barrier that is provided toallow for rapid loading of the wells and for rapid sealing from the bulkfluid. In another example, combined chemical and physical barriers areused, wherein the physical barrier is used to seal the wells and thenthe chemical barrier conditionally releases the oligonucleotide primersinto the well solution, for example by heating, slow release, orenzymatic digestion. Well depth or channel length to each well also maybe used to control release of the reagents from the wells. Other meansfor loading high density array 581 are possible.

FIGS. 15-17 show an illustrative embodiment of second-stage 580 using aphysical barrier. Sandwiched between first layer 518 and second layer519 of pouch 510 is high density array 581, with wells 582. As best seenin FIG. 16, pierced layer 585, with piercings 586, is provided on oneside of high density array 581 to act as the physical barrier, and asecond layer 587, is provided on the opposite side of high density array581 to form the bottom of wells 582. Illustratively, pierced layer 585and second layer 587 are plastic films that have been sealed to highdensity array 581, illustratively by heat sealing, although it isunderstood that other methods of sealing may be employed. It is alsounderstood that the material used for high density array 581 and thematerial used for pierced layer 585 and second layer 587 should becompatible with each other, with the sealing method, and with thechemistry being used. When used for PCR, examples of compatible plasticsthat can used for high density array 581 and can be heat-sealed are PE,PP, Monprene®, and other block copolymer elastomers. If fluorescent dyesare used in the detection chemistry, it may be desirable for highdensity array 581 to be formed from black or other relativelyfluorescently opaque materials, to minimize signal bleed from one well582 to its neighboring wells and for at least one of layers 585 and 587to be relatively fluorescently transparent. For pierced layer 585 andsecond layer 587, laminates of a strong engineering plastic such asMylar® or PET with heat-sealable plastic layers such as PE, PP andDupont Surlyn® may be used. For adhesive-based systems, rigidengineering plastics such as PET or polycarbonate may be used to formhigh density array 581 and films of PCR-compatible plastics are thenused as pierced layer 585 and second layer 587. In one illustrativeembodiment, high density array 581 is formed of black PE, a compositepolyethylene/PET laminate (or Xerox® PN 104702 hot laminating pouchmaterial) is used for pierced layer 585 and second layer 587 which areheat sealed to high density array 581, and composite polypropylene/PETis used for first and second layers 518, 519 of pouch 510.

It is understood that piercings 586 align with wells 582. It is alsounderstood that piercings 586 are small enough that, absent some force,fluid does not readily flow through piercings 586. Illustrativepiercings may be 0.001-0.1 mm, more illustratively 0.005-0.02 mm, andmore illustratively about 0.01 mm. In the illustrative embodiment,second-stage amplification zone 580 is provided under vacuum, such thatwhen fluid is received from blister 566, the vacuum draws fluid throughpiercings 586 into each well 582. Once the wells 582 are filled, a forceis no longer present to force fluid into or out of the wells 582. Abladder adjacent second-stage amplification zone 580 (not shown, butsimilar in position to bladders 880/882) may then be activated to pressfirst layer 518 against high density array 581 and seal fluid into thewells 582. While first layer 518 of pouch 510 is used to seal the wells582, it is understood that an optional sealing layer may be providedbetween pierced layer 585 and first layer 510.

In one illustrative example, second-stage amplification zone 580 may beprepared as follows. High density array 581 may be prepared by firstpunching, molding, or otherwise forming an array of wells 582 in aplastic sheet (illustratively 0.1 to 1 mm thick). The wells may form anyregular or irregular array that is desired, and may have a volumeillustratively of 0.05 μl to 200, and more illustratively of 0.1 μl to 4μl. One of layers 585 or 587 is then laminated to a first surface 581 aof high density array 581, illustratively by heat or adhesive. As shownin FIG. 17, pierced layer 585 is applied to first surface 581 a.Reagents 589, illustratively elements of the chemistry of the array thatare unique, such as PCR primer pairs, are then spotted into the wellseither manually by pipetting, or automatically (illustratively using x/ypositionable spotters such as pin-spotters, dot-matrix printers,small-volume automatic pipettes, or micro-fluidic micro-contactspotters). After the reagents 589 have been dried in each well 582, thesecond of layers 585 or 587 is applied to the second surface 581 b ofarray 581. Layer 585 is pierced using an array of small diameter needlesto form piercings 586. Piercings 586 may be formed either before orafter layer 585 has been fixed to array 581. It is understood thatspotting can be done on either layer 585 before or after piercing or onlayer 587. Spotting an array with holes pre-pierced has not shown toleak substantially and offers the advantage that the needles used forpiercing are not contaminated by touching the spotted reagents.Alternatively, to minimize the possibility of leakage, and to positionthe spotted reagents at the most distant location in the array, it maybe desirable to spot the reagents 589 onto second layer 587, seal thearray 581 with layer 585, and then pierce layer 585. In an illustrativeexample, reagents are spotted onto second layer 587 using a GeSiMA060-324 Nano-Plotter 2.1/E (Grosserkmannsdorf, Germany) Using such aspotter, multiple arrays may be spotted simultaneously.

Once spotted and pierced, array 581 is placed inside layers 518 and 519of pouch 510 and sealed in place, illustratively by either heat sealing,using an adhesive, ultrasonically welding, mechanical closure, or othermeans of enclosing array 581 inside pouch 510 within blister 584. It isunderstood that blister 584 is fluidly connected to blister 566 viachannel 565, and that liquid can flow from channel 565 into blister 584and over piercings 586. In one illustrative example, when blister 584 isformed, care is taken to allow a path for air to escape. This can beaccomplished by “waffling” the inside surface of first layer 518adjacent to second-stage amplification zone 580 to imprint the filmmaterial with a pattern of slightly raised texture. This allows air andliquid to pass along the surface of pierced layer 585, and better allowsliquid to reach and fill all of wells 582. The pouch 510 is then placedinside a vacuum chamber and evacuated. Illustratively, when the pressurehas reached approximately 0.3 millibars, a pneumatic cylinder inside thevacuum chamber is actuated, driving down a plunger into fitment 590 toseal channel 567, thereby cutting the path from the array inside thesealed pouch, and the vacuum chamber. A plurality of other plungers arealso driven into fitment 590 to seal the various entry channels 515. Thepouch is removed from the vacuum chamber and may be packaged forlong-term storage in a vacuum-bag.

Pouch 510 may be used in a manner similar to pouch 210. Because array581 is packaged in vacuum, when liquid is moved from blister 566 tosecond-stage amplification zone 580, the liquid sample is drawn throughpiercings 586 and into wells 582. Excess liquid is forced away byinflating a pneumatic bladder over the array and thermal cycling isaccomplished as described above, illustratively by heating and cooling aPeltier element pressed against one side of the array.

As mentioned above, pierced layer 585 may be replaced by a variety ofsuitable physical or chemical barriers. In one illustrative embodimentusing a chemical barrier, pierced layer 585 is omitted, and reagents 589are spotted into wells 582 in a buffer that dissolves relatively slowly.Illustratively, reagents 589 that contain polymers such as PEG, Ficollor polysorbate 20 or sugars such as sucrose, trehalose or mannitol inappropriate concentrations will be compatible with the second-stage PCRreaction and may dissolve more slowly than primers spotted solely inwater or Tris/EDTA. The primers spotted in one of these buffers may beair dried into the wells 582, as described above (it is understood thatin such an embodiment, second layer 587 is affixed to high density array581 for spotting). These same polymers may be used in lyophilization ofenzyme reagents (e.g. the enzymes and buffers used in PCR) to form anopen matrix containing the stabilized enzymes. Thus, the primers spottedin these buffers can be lyophilized in place in the wells 582, leadingto slower but potentially more complete rehydration than with airdrying. When pouch 510 is used, the fluid from blister 566 is driveninto the well by vacuum or pressure and starts to dissolve the primermix. By selecting a buffer that dissolves suitably slowly, when thebladder adjacent second-stage amplification zone 580 is actuated, thecontents of each well 582 is sealed therein prior to any substantialcross-contamination.

Another embodiment uses a matrix that does not dissolve untilsecond-stage amplification zone 580 is heated above a predeterminedtemperature. One example of such a matrix is low melt agarose such asGenePure LowMelt Agarose (ISC Bioexpress). In one example, a 1.5%solution of this agarose melts at 65° C. and gels at 24-28° C. Prior tospotting, reagents 589 illustratively may be warmed to 50° C. and mixedwith this agarose that had already been melted and then spotted intowells 582 in a small volume (illustratively 100 to 500 nl). To keep themixture liquid during spotting, this may have to be done in a cabinetheated above the melting temperature of the agarose. Alternatively, itmay be possible to pipette dilute solutions of the agarose withoutmelting. After the agarose/reagent mixture is spotted, the high densityarray 581 is dried. This can be a simple air drying or theprimer-agarose mixture can contain the sugars and polymers listed aboveso that the reagents can be freeze dried. When pouch 510 is used forPCR, second-stage amplification zone 580 may be heated, illustrativelyto 55° C., as the fluid from blister 566 is moved into high densityarray 581. At this temperature, the agarose does not melt so the primersare not released into solution. Once high density array 581 is filled,the corresponding bladder is inflated to seal the wells. When thetemperature rises above 65° C. in the first denaturation step of thefirst PCR cycle, the agarose containing the primers melts, releasing theprimers into the master mix. Illustratively, thermal cycling never goesbelow 60° C. (or other melting temperature for the agarose) so that theagarose does not gel during thermal cycling. Furthermore, in theillustrative instrument 800 of FIG. 8, the repeated temperature cyclingis driven by heater 888, which is located on one side of the pouch. Itis expected that there will often be a temperature gradient across thePCR solution in wells 582, which should facilitate mixing of the primersby convective fluid flow. Wax may also be used in a similar embodiment.

In a further embodiment, the primers may be conditionally bound to thewells 581, with subsequent releasing of the primers into solution afterthe wells 581 have been filled. Depending upon how the primers areattached to the plastic substrate, the primers may be cleaved using heat(illustratively during the first cycle of the PCR reaction), light(illustratively irradiating through window 847), chemicals (e.g.dithiothreitol together with heat will reduce disulfide bonds that maybe used to link primers to the wells), or enzymes (e.g. site specificproteases such at Tissue Plasminogen Activator can be used to cleave theproper peptide linker attaching primers to the substrate).

In yet another embodiment, a DNase may be injected into second-stageamplification zone 580 subsequent to amplification, to minimize furtherany potential risk of contamination.

It is understood that second-stage amplification zone 580 has beendescribed herein for use with PCR. However, other uses for pouch 510 andsecond-stage amplification zone 580 are within the scope of thisinvention. Further, it is understood that second-stage amplificationzone 580 may be used with or without nucleic acid extraction and a firststage PCR amplification zone. Finally, it is understood thatsecond-stage amplification zone 580 may be used with any of the pouchembodiments described herein.

Example 1: Nested Multiplex PCR

A set of reactions was run in a pouch 110 of FIG. 5, on an instrumentsimilar to instrument 800 but configured for pouch 110. To show celllysis and effectiveness of the two-stage nucleic acid amplification, 50μL each of a live culture of S. cerevisiae and S. pombe at log phase wasmixed with 100 μL of a nasopharyngeal aspirate sample from a healthydonor to form the sample, then mixed with 200 μL lysis buffer (6Mguanidine-HCl, 15% TritonX 100, 3M sodium acetate. 300 μL of the 400 μLsample in lysis buffer was then injected into chamber 192 a of pouch110.

The pouch 110 was manufactured with 0.25 g ZS beads sealed inthree-lobed blister 122. Second-stage primers, as discussed below, werealso spotted in blisters 181 and 182 during manufacture of pouch 110.The pouch 110 was loaded as follows:

115 a sample and lysis buffer, as described above,

115 b magnetic beads in the lysis buffer,

115 d-e wash buffer (10 mM sodium citrate),

115 g elution buffer (10 mM Tris, 0.1 mM EDTA)

115 h first-stage PCR buffer:

-   -   0.2 mM dNTPs    -   0.3 μM each primer:        -   Sc1: primers configured for amplifying a portion of the YRA1            nuclear protein that binds to RNA and to MEX67p of S.            cerevisiae. The primers are configured to amplify across an            intron such that amplification of cDNA (mRNA            reverse-transcribed via M-MLV) yields a 180 bp amplicon.        -   Sc2: primers configured for amplifying a 121 bp region of            the cDNA of MRK1 glycogen synthase kinase 3 (GSK-3) homolog            of S. cerevisiae.        -   Sc3: primers configured for amplifying a 213 bp region of            the cDNA of RUB1 ubiquitin-like protein of S. cerevisiae.        -   Sp1: primers configured for amplifying a 200 bp region of            the cDNA of suc1-cyclin-dependent protein kinase regulatory            subunit of S. pombe.        -   Sp2: primers configured for amplifying a 180 bp region of            the cDNA of sec14-cytosolic factor family of S. pombe.    -   PCR buffer with 3 mM MgCl₂ (without BSA)    -   50 units M-MLV    -   4.5 units Taq:Antibody    -   100 units RNAseOut

115 j-k second-stage PCR buffer

-   -   0.2 mM dNTPs    -   1× LC Green® Plus (Idaho Technology)    -   PCR buffer with 2 mM MgCl₂ (with BSA),    -   4.5 units Taq

115 l second-stage PCR buffer with a sample of the first-stageamplicons.

During manufacture, second-stage blisters 181 and 182 were spotted withnested second-stage primers. Each blister was spotted with one primerpair in an amount to result in a final concentration of about 0.3 μMonce rehydrated with the second-stage PCR buffer. The second-stagenested primers are as follows:

-   -   Sc1: primers configured for amplifying an 80 bp fragment of the        Sc1 cDNA first-stage amplicon.    -   Sc2: primers configured for amplifying a 121 bp fragment of the        Sc1 cDNA first-stage amplicon.    -   Sc3: primers configured for amplifying a 93 bp portion of the        Sc1 cDNA first-stage amplicon.    -   Sp1: primers configured for amplifying a 99 bp portion of the        Sc1 cDNA first-stage amplicon.    -   Sp2: primers configured for amplifying a 96 bp portion of the        Sc1 cDNA first-stage amplicon.        There is no overlap between the first-stage and second stage        primer pairs for any of the targets. Each pair of primers was        spotted into one negative control blister 181 and two        second-stage blisters 182, so that each second-stage        amplification would be run in duplicate, each duplicate with a        negative control.

After loading, activation of the plunger associated with entry channel115 a moved the sample to three-lobed blister 122, activation of theplunger associated with entry channel 115 b moved the magnetic beads toreservoir 101, activation of the plungers associated with entry channels115 d-e moved wash buffer to reservoirs 102 and 103, activation of theplunger associated with entry channel 115 g moved elution buffer toreservoir 104, activation of the plunger associated with entry channel115 h moved first-stage PCR buffer to reservoir 105, activation of theplungers associated with entry channels 115 j-k moved second stage PCRbuffer to reservoirs 106 and 107, and activation of the plungerassociated with entry channel 115 l moved the positive control(second-stage PCR buffer with a sample of previously preparedfirst-stage amplicon) to reservoir 108. In this present example, theplungers associated with entry channels 115 a and 115 b were depressedprior to loading the pouch 110 into the instrument. All other plungerswere depressed sequentially in the instrument during the run, and fluidswere moved to reservoirs 102 through 108 as needed.

Once pouch 110 was placed into the instrument, and beating took placefor ten minutes in the presence of ZS beads, as described above. Oncecell lysis was complete, reservoir 101 was compressed and nucleic acidbinding magnetic beads from reservoir 101 were forced into three-lobedblister 122, where the beads were mixed gently and allowed to incubatefor 5 minutes.

The sample-bead mixture was then moved to blister 144, where themagnetic beads were captured via activation of the magnet. Once themagnet was deployed, bladders adjacent blister 144 were pressurized toforce fluids back to three-lobed blister 122. The captured beads werethen washed as described above, using the wash solution from reservoirs102 and 103. Following washing, the beads were once again captured inblister 144 via activation of the magnet, and the elution buffer storedin reservoir 104 is moved to blister 144, where, after a 2 minuteincubation, the nucleic acids eluted from the beads are then moved toblister 161, as discussed above.

In blister 161, the nucleic acid sample is mixed with first-stage PCRmaster mix from reservoir 105. The sample is then held at 40° C. for 10minutes (during which time M-MLV converts mRNA to cDNA), then 94° C. for2 minutes (to inactivate the M-MLV and remove antibody from taq).Thermal cycling is then 20 cycles of 94° C. for 10 second and 65° C. for20 seconds.

Subsequent to first-stage amplification, the sample is dilutedapproximately 100-fold using the second-stage PCR master mix fromreservoir 106. The sample is then moved to blisters 182, which werepreviously spotted with the second-stage primers, as discussed above.Second-stage PCR buffer was moved from reservoir 181 to negative controlblisters 181, and the positive control mixture was moved to blisters 183from reservoir 108. The samples were denatured for 30 seconds at 94° C.,then amplified for 45 cycles of 94° C. for 5 seconds and 69° C. for 20seconds.

As can be seen in FIG. 13, all target amplicons and the positive controlshowed amplification, while none of the negative controls showedamplification. Each sample was run in replicates. The replicates eachshowed similar amplification (data not shown).

It is understood that the S. cerevisiae and S. pombe targets areillustrative only and that other targets are within the scope of thisinvention.

Example 2: High Density PCR

The above example uses pouch 110 of FIG. 5. Pouch 110 has five negativecontrol blisters 181, five positive control blisters 183, and ten lowvolume sample blisters 182. Pouch 210 of FIG. 6 increased the number oflow volume sample blisters 282 to 18. However, high density array 581 ofpouch 510, shown in FIG. 15 can have 120 or more second-stage wells 582.This increase in the number of second-stage reactions enables a wide setof potential diagnostic and human identification applications withoutthe need to increase the size of the pouch and its instrument. Variousexamples are described herein.

In one example, it is known that standard commercial immunofluorescenceassays for the common respiratory viruses can detect seven viruses:Adenovirus, PIV1, PIV2, PIV3, RSV, Influenza A, and Influenza B. A morecomplete panel illustratively would include assays for additional fiveviruses: coronavirus, human metapneumovirus, BOCAvirus, Rhinovirus andnon-HRV Enterovirus. For highly variable viruses such as Adenovirus orHRV, it is desirable to use multiple primers to target all of thebranches of the virus' lineage (illustratively 4 outer and 4 innerprimer sets respectively). For other viruses such as coronavirus, thereare 4 distinct lineages (229E, NL63, OC43, HKU1) that do not vary fromone season to another, but they have diverged sufficiently enough thatseparate primer sets are required. The illustrative complete respiratoryvirus panel would also target the SARS coronavirus, possibly the avianinfluenza HA and N subtypes, and possibly others. Finally, some of therespiratory viruses show such a high rate of sequence variation that itwould be beneficial to create more than one nested PCR assay for eachsuch virus, thereby minimizing the chance of false negative results dueto sequence variation under the primers. When all of the primer setsdescribed herein are included, such a respiratory virus panel could have80 or more specific amplicons in the second-stage amplification. Thehigh density array 581 could easily accommodate such a panel in a singlepouch 510.

A second application of the high density array 581 of pouch 510 would beto determine the identity and the antibiotic resistance spectrum of themulti-drug resistant bacteria isolated from infected patients. Currentmethods require several days to culture the organism and empiricallytest individual drug resistance profiles. During the time it takes toreceive the results, physicians will often administer broad-spectrumantibiotics, which leads to an increase in multi-drug resistantbacteria. PCR primers have been developed to detect the geneticdeterminants of antibiotic resistance (the antibiotic resistance genesthemselves). However because of the large number of variants of some ofthese genes, a large number of amplicons is required for a completedetermination of the resistance profile. Hujer et. al. describe a panelof 62 PCR assays to identify the resistance genes present inAcinetobacter isolates. Again, the high density array 581 could easilyaccommodate such a panel in a single pouch.

A third example of the utility of the high density array is in the fieldof human identification, illustratively for forensic identification ofhuman remains and for paternity testing. Most of the market in humanidentification is dominated by systems that analyze short tandem repeatsequences (STRs). This analysis has generally required separating therepeats by size, using e.g. capillary electrophoresis. The specializedlaboratory equipment used for this purpose has generally not been fieldportable. There is growing interest in using Single NucleotidePolymorphisms (SNPs) for identity testing, as there are a large set oftechniques for identifying SNPs and some of these are amenable to fielduse. Sanchez et al. have published a set of 52 well-characterized SNPsthat collectively give a very low probability of matching twoindividuals by chance (a mean match probability of at least 5.0×10⁻¹⁹).In practice, it may take two amplicons for each SNP to accurately typeeach locus (see, e.g., Zhou et al.). Thus one pouch 510 with 104second-stage wells 582 could completely type an individual at all of the52 SNP loci.

It is understood that there are cost and workflow advantages gained bycombining assays from different diagnostic applications into one pouch.For example the complete respiratory virus panel could be combined withthe bacterial identification panel. These combinations could simplifymanufacturing, since there are fewer types of pouches to assemble. Theycould also simplify the work of the end user, as there are fewerspecific types of pouches that need to be stocked in a clinic, and alsoreducing the chance of using the wrong pouch for a particular clinicalsample. For some applications, these advantages could offset an increasecost of manufacturing the pouch having a greater number of primer pairs.Thus one pouch 510 with 100 or more second-stage wells 582 could be usedto accommodate multiple panels of assays.

Example 3: Process Controls

Controls for highly multiplexed assays can be problematic, especially inclinical diagnostic settings where quality must compete with cost pertest. The high-density array 582 of pouch 510 potentially increases thisproblem because of the increased number of diagnostic targets that canbe assayed in a single run. Various types of controls are discussedherein.

Illustrative process controls include mixing an intact organism, forexample an organism containing an RNA target, into the patient samplebefore injecting the sample into the pouch. Such a target could be anintact RNA bacteriophage (MS2 or Qβ) or an intact plant RNA virus(Tobacco Mosaic Virus) or an mRNA present in an intact yeast. Outerprimers specific for the RNA target would be present in the first-stagePCR and a well 582 containing the inner primers would be present in thehigh density array. Detection of amplification product in this well 582confirms that all of the steps of the process are working correctly. Apost-second-stage amplification melt curve could also be used to verifythat the correct specific product was made. The crossing point (“Cp”)determined from an amplification curve could be used to give aquantitative measure of the integrity of the reagents. For example theCp can be compared to that of other pouches from the same lot run at adifferent time. While an intact organism is used, it is understood thatpurified or isolated nucleic acids may be used if it is not important totest for lysis. In other situations, it may be desirable to use thecontrol to test only the later steps of the analysis. For example,spiking a natural or synthetic nucleic acid template into a well in thehigh density array along with the cognate primers could be used to testthe second-stage PCR reaction, and spiking a nucleic acid template intothe first-stage PCR with the appropriate primers in the first-stage PCRamplification mixture and in a well 582 of the second-stageamplification zone will test both the first- and second-stage PCRreactions.

Process controls such at described above do not test the integrity ofthe primers specific to the target amplicons. One example of a positivecontrol that tests the integrity of the specific primers uses a mixtureof nucleic acids, illustratively synthetic RNAs, as stability andvariability often can be better controlled and these sequences cannot bepresent due to environmental contamination, wherein the mixture containsa nucleic acid for each of the primers present in the particular pouch.In a diagnostic setting, this positive control could be used at the endof a run of pouches used to test patient samples. The mixture isinjected into a pouch, illustratively from the same lot as those usedfor the patient samples, and success is defined by all of the targetamplicons providing a positive result. Negative controls can be done inthe same way; at the end of a run of pouches used to test patientsamples, water or buffer could be injected into a pouch and successdefined by all of the target amplicons providing a negative result.

Individual workflow and protocols in a diagnostic lab may be used todetermine the number of patient sample pouches run before the controlpouches described above are run. Regardless of how frequently orinfrequently the control pouches are run, these controls add to the timeand cost of the total system. For this reason, it would be useful tomake the controls internal to the pouch. The structure of the highdensity array 581 allows for the following novel approach to negativecontrols. In this example, a nucleic acid, illustratively a syntheticamplicon, is spiked into one of the wells 582 a of the high densityarray 581. Primers to amplify this sequence are spiked into this well582 a and into two other wells 582 b and 582 c spaced across the array.Illustratively, the amplicon sequence and primers are artificial anddesigned so that none of the primers used will amplify another target bychance.

When a clean, uncontaminated pouch 510 is run in instrument 800, thewell 582 a containing the synthetic target will generate amplicon andtherefore be called positive. The two other wells 582 a, 582 b thatcontain the corresponding primers should not amplify anything in thesample and thus be called negative. Pouch 510 may be treated further,for additional controls. Illustratively, bladder 880/882 holding thehigh density array against heater 888 is then depressurized and thecontents of the wells 582 are mixed. In one illustrative method, thecontents of the wells 582 are mixed as follows: heater 888 is used tocycle the temperature of the high density array above and below theboiling point of the buffer for a short time (for example three cyclesof 85° C. for 10 sec then 105° C. for to 20 sec). Bubbles of steamgenerated in the wells 582 of high density array 581 should force thecontents of wells 582 out into the second-stage amplification zoneblister 580. Optionally, the contents of the second-stage amplificationzone 580 may be mixed with the contents of rest of the pouch 510 byusing the bladders to move liquid from one end of the pouch 510 to theother. The purpose of these steps is to mix the specific contaminationcontrol amplicon, along with any specific target amplicons throughoutthe pouch.

If the user accidentally opens a pouch after it has been run in thisfashion, then both specific target amplicons and the contaminationcontrol amplicon will be released. If trace amounts of these nucleicacids contaminate a later pouch run, the instrument may detect thecontamination event, as the wells 582 b, 582 c that contained only theprimers specific for the synthetic amplicon will score positive.Software in the instrument will alert the user and the results of therun will be flagged as suspect.

In another method to control contamination, at the end of a run, a DNAdegrading chemical or enzyme may be added to destroy substantially allof the DNA products of the first- and second-stage PCR reactions.Illustratively, this can be done in a way similar to the contaminationdetection method described above, by heating the contents of thesecond-stage array to above the local boiling temperature, thus drawingthe amplified sample out of the wells 582 of the array 851, mixing theheated liquid with the diluted contents of the 1^(st) stage reaction,adding an aliquot of a DNA degrading substance, illustratively throughentry channel 515 k, either with or without cooling the mixture, andallowing the DNA degrading reaction to incubate until substantially allof the DNA produced in the PCR reaction has been destroyed. This can beaccomplished using DNAases, acids, or oxidants, as are known in the art.

It is understood that any of the contamination controls described hereinmay be used independently or in any combination thereof.

Example 4: iPCR

In another example, the pouches and instruments of the present inventionmay be used for immuno-PCR (iPCR). iPCR combines the antibodyspecificity of ELISA with the sensitivity and multiplex capabilities ofPCR. While iPCR has been applied to diagnostics and toxin detection,iPCR has not enjoyed widespread commercial application, presumablybecause PCR template contamination issues are severe in an open ELISAformat. Because the pouch format of the present invention provides asealed environment, the pouches of the present invention may be wellsuited for iPCR.

A traditional ELISA detection scheme is shown in FIG. 11 (labeled“ELISA”). In 1992, Cantor and colleagues (Sano, T., et al, Science,1992. 258(5079): p. 120-2, herein incorporated by reference) described amodification of the basic ELISA technique (FIG. 11, similar to the“Immuno-PCR I” scheme without capture antibody C-Ab), in which theenzyme used for generating a specific signal is replaced by a unique DNAfragment indirectly attached to the reporter antibody R through abi-functional binding moiety S, such as a streptavidin-protein Achimera. The DNA fragment is subsequently detected by PCR. It is knownthat PCR detection can provide dramatic increases in immuno-PCR assaysensitivity over corresponding ELISA assays, with improvements tosensitivity commonly 10² to 10⁴-fold. Advances in quantitative real-timePCR methods have improved the speed and quantification of immuno-PCR.Direct coupling of the reporter antibody (R-Ab) with DNA template tags(FIG. 11, “Immuno-PCR II” scheme) has further increased the assaysensitivity 10² to 10³ fold and made possible the development ofmultiplex immuno-PCR assays, in which each different antibody is taggedwith a different oligonucleotide and, thus, each antigen is associatedwith a unique amplification product.

Despite these advantages over traditional ELISAs, iPCR has not beenwidely adopted in commercial products in the 13 years since it was firstdescribed. This is due in part to the contamination hazards inherent inany open-tube PCR analysis method. Prior art iPCR protocols are derivedfrom ELISA assays and require numerous wash steps that increase thelikelihood of contaminating the work area with amplified material. Thesignificant risk of false positives due to workflow contamination hascontributed to the avoidance of iPCR in diagnostic assessment.

Amplicon contamination issues slowed the widespread adoption of PCRitself in the diagnosis of human genetic conditions or of infectiousdisease until homogenous (i.e. “closed-tube”) PCR assays were developed.By making the readout of the assay possible in a closed-tube system,spread of amplicon is severely curtailed. Similarly, iPCR may be morewidely adopted if a closed system format were available. In the presentsystem, the sample would be injected into a pouch that would be providedwith all required reagents. The steps of antigen capture, wash,reporter-antibody binding, wash, and subsequent PCR detection could beperformed completely within the pouch. Illustratively, nucleic acidswould never leave the pouch and would be disposed of along with thepouch.

Any of the pouches of the present invention may be adapted for iPCR. Forexample, the pouch 210 of FIG. 6 illustratively may be adapted asfollows. Chambers 292 a through 292 l would be filled with the followingcomponents. The sample illustratively comprising an unpurified and/orunmodified antigen (e.g. a toxin) is injected through injection port 241a to chamber 292 a. A capture antibody conjugated to magnetic beads(C-Ab) is provided in chamber 292 b. If multiple targets are to betested, it is understood that multiple capture antibodies havingspecificity for multiple antigens may be used. An optional pre-washbuffer is provided in chamber 292 c. A reporter antibody conjugated toan oligonucleotide template (R-Ab-DNA) is provided in chamber 292 d. Itis understood that the capture and reporter antibodies may be monoclonalor polyclonal. When multiple antigens are to be detected, the captureand reporter antibodies may contain only polyclonal antibodies, onlymonoclonal antibodies, or any combination of polyclonals specific forone antigen and monoclonals specific for another antigen. When areporter antibody is polyclonal, it is understood that all reporterantibodies having specificity for a particular antigen will be coupledto oligonucleotide templates have one specific sequence, even if thespecificity between various antibodies in that set varies. Theoligonucleotide may be double-stranded or single-stranded. MultipleR-Ab-DNAs may be provided to detect multiple antigens, with eachdifferent antibody conjugated to a unique oligonucleotide. Wash buffersare provided in chambers 292 e through 292 h. A first-stage PCR mastermix, as described above, is provided in chamber 292 i. A dilution bufferis provided in chamber 292 j. A second-stage PCR master mix, asdescribed above, is provided in chamber 292 k. As discussed above, thereagents may be provided dried in chambers 292 b through 292 l, and maybe rehydrated prior to use via injection of water through seal 239, oreach reagent may be provided wet via injection to each individualchamber 292. Combinations thereof are contemplated.

Once the sample and reagents are loaded, the pouch 220 is inserted intothe instrument 800. Plunger 268 a is then depressed and the sample ismoved to three-lobed blister 222. Plunger 268 b is also depressed andthe capture antibodies conjugated to magnetic beads (shown as C in FIG.11) are also moved to three-lobed blister 222. The sample and the C-Abare mixed via pressure from bladder 828 alternating with pressure frombladders 824, 826. Because mixing is desired, pressure from the bladders824, 826, 828 may be considerably lower than the pressure used asdiscussed above for lysis. Illustratively, gentle mixing is obtained.The sample and the C-Ab are allowed to incubate for sufficient time forthe capture antibodies to bind to antigens T in the sample (formingC-Ab-T complexes), illustratively for about 5 minutes, although otherincubation times may be desirable. For iPCR, it may be desirable toinclude an additional heater in instrument 800 to maintain incubationsat about 37° C.

Once the antigens present in the sample have been sufficiently incubatedfor capture, the sample is moved to blister 246 and the magnet 850 isdeployed, capturing the capture antibodies in blister 246. The unboundportions of the sample are then moved back to three-lobed blister 222,which now functions as a waste reservoir. While magnetic beads are usedto restrain the capture antibodies in the examples described herein, itis understood that other capture mechanisms may be used, including solidsupports, possibly even cross-linking the capture antibodies to aninterior surface of a blister.

If desired, the C-Ab-T complexes may be washed using the pre-wash bufferfrom chamber 292 c. Pre-wash buffer is moved into blister 244 viachannel 245, the magnet is withdrawn, releasing the C-Ab-T complexes,and the beads are gently moved between blisters 244 and 246. The beadsare then recaptured in blister 246 via activation of the magnet 850, andthe remaining fluid is moved to three-lobed blister 222. It is expectedthat this pre-wash may improve discrimination of a positive signal overthe background negative signal, but such differences may prove to beinsignificant. Additional pre-washes may be performed, if desired.

Plunger 268 d is depressed and the mixture containing one or morereporter antibodies R-Ab conjugated to oligonucleotide templates(R-Ab-DNA, shown in FIG. 11, Immuno-PCR II scheme, as R with attachednucleic acid) is moved to blister 246. The magnet is retracted and themixture is gently mixed by moving between blisters 244 and 246.Incubation, illustratively for about 5 minutes although other incubationtimes may be desirable, allows formation of the ternary complexesC-Ab-T-R-Ab-DNA, as illustrated in FIG. 11, Immuno-PCR II. Activation ofthe magnet 850 allows capture of the ternary complexes in blister 246,and the remaining fluid is moved to three-lobed blister 222.

Plunger 268 e is depressed and wash buffer is moved from chamber 292 eto blister 246. The magnetic bead-ternary complex is washed as in thepre-wash described above, the magnetic bead-ternary complex isrecaptured in blister 246, and the remaining fluid is moved tothree-lobed blister 222. Washing is repeated multiple times using thewash buffers from chambers 292 f, 292 g, and 292 h, except that mixingis between blisters 246 and 248 to avoid reintroducing unbound R-Ab-DNAcomplexes that may be residing in blister 244 or channel 243. While fourwashes are described in this illustrative embodiment, it is understoodthat any number of washes may be used, illustratively by altering thenumber of chambers in the fitment 290 or by increasing the volume of thechambers and using only a portion of the wash buffer in a chamber foreach wash. It is also understood that removal of all unbound R-Ab-DNAcomplexes is extremely difficult, even with a large number of washes.Further, for an antigen that is not present in the sample, the presenceof just a few molecules of unbound R-Ab-DNA or non-specifically boundR-Ab-DNA complexes specific for that antigen may result in anamplification signal. Thus, while the ideal goal of the washing step isto remove all R-Ab-DNA complexes specific for antigens that are notpresent in the sample, one illustrative goal is to remove a sufficientnumber of such R-Ab-DNA complexes such that the amplification curve forthat oligonucleotide is delayed and can be distinguished from theamplification curve of a positive sample. Illustratively, more washesshould remove more unbound R-Ab-DNA and provide for a lower detectionlimit, but more washes risk loss of desired ternary complexes throughdissociation or loss of magnetic beads not captured by the magnet. Afterwashing is complete, if desired, the captured ternary complex may beheated or enzymatically treated (illustratively with papain, proteinaseK, or other suitable enzyme provided via an additional chamber) torelease the DNA prior to PCR. Such treatment may improve the first-stagePCR efficiency. It is understood that such treatment may be used withany of the iPCR examples discussed herein.

Once washing is complete, plunger 268 i is depressed and the first-stagePCR master mix, as described above, is moved to blister 246. First-stagePCR master mix contains primer pairs for all desired targets. The magnet850 is released, and optional mixing between blisters 246 and 248 may beused to resuspend the ternary complexes. The mixture is moved to blister264, where first-stage thermal cycling takes place, as described above.Once the complexed oligonucleotides have been amplified to sufficientlevels, as discussed above, the amplified mixture is optionally dilutedusing the dilution buffer provided in chamber 292 j. Some or all of thefirst-stage amplified mixture may be mixed with the second-stage PCRmaster mix provided from chamber 292 k, and then this mixture is movedto the 18 second-stage blisters 282, where second-stage primers areprovided, as discussed above. If desired, one of the second-stageblisters 282 may be used for a negative control, wherein it is knownthat no antigen is present in the sample, but R-Ab-DNA was provided fromchamber 292 d and the proper primers are provided in the negativecontrol second-stage blister 282. It is expected that, despite variouswashes, small amounts of this particular R-Ab-DNA may be present in thefirst-stage PCR and, accordingly, that small amounts of the first-stageamplified product may be provided to this second-stage blister 282.However, the amounts should be quite small, and the crossing pointshould be delayed well past that of positive samples. Also, if desired,one of one of the second-stage blisters may be used for a positivecontrol, wherein the sample is spiked with an antigen that is nototherwise being tested (perhaps included with the C-Ab beads), whichpresumably will bind its corresponding R-Ab-DNA, and which is thenamplified in the first-stage PCR. Finally, control blisters 283 are notused in this illustrative embodiment. However, with a minorreconfiguration, blisters 283 may be connected to blister 266 and mayprovide for six additional second-stage reactions. Alternatively,blisters 283 may be used for other controls, as are desired by theparticular application.

As discussed above, because of the difficulty in removing all unbound ornon-specifically bound R-Ab-DNA complexes, even negative samples mayshow some amplification. It is expected that real-time amplificationanalysis will allow positives to be distinguished from negatives via adifference in cycle number of a threshold crossing point (or anequivalent cycle threshold measurement, such as the cycle number when50% of amplification is reached).

It is understood that the first-stage multiplex amplification may not benecessary for detection with iPCR, even when testing for multipleantigens. However, the first-stage multiplex amplification may affordmore sensitivity.

Example 5: iPCR with iPCR-Specific Pouch

The above example illustrates a method adapting the pouch 210 of FIG. 6for iPCR. However, FIG. 12 shows a pouch 310 that is illustrativelyconfigured for iPCR. Fitment 390 is similar to fitments 190 and 290,except having 15 chambers 392 and plungers 368. Each chamber 392(illustratively chamber 392 a, where the sample is injected) may haveits own injection port, or several chambers may have a connectingchannel and may share an injection port (illustratively 392 e through392 k, each containing wash buffer). As with the above-describedfitments, any combination of injection ports and channels is within thescope of this invention. Pouch 310 differs from pouch 210 of FIG. 6 inone primary way. As cell lysis is usually not needed in iPCR, thethree-lobed blister 222 may be replaced by a single large wastereservoir 322. Because multiple washes are desirable in iPCR, wastereservoir 322 is provided with a sufficiently large volume to retain themultiple used buffers, for example 2-5 ml, depending on the applicationand volume of the reactions. It is understood that instrument 800 mayneed to be reconfigured somewhat to accommodate pouch 390.

Prior to insertion into the instrument, pouch 390 of FIG. 12illustratively would have the following components in the chambers 392.The sample to be tested would be injected into chamber 392 a. Captureantibodies (C-Ab) conjugated to magnetic beads are provided in chamber392 b. An optional pre-wash buffer is provided in chamber 392 c.Reporter antibodies conjugated to their respective oligonucleotidetemplates (R-Ab-DNA) are provided in chamber 392 d. As discussed above,multiple R-Ab-DNAs may be provided to detect multiple antigens, witheach different antibody conjugated to a unique oligonucleotide. Washbuffers are provided in chambers 392 e through 392 k. A first-stage PCRmaster mix is provided in chamber 392 l. A dilution buffer is providedin chambers 392 m and 392 n. A second-stage PCR master mix is providedin chamber 392 o.

To begin, plungers 368 a and 368 b are depressed, forcing the sample andthe capture antibodies C-Ab through channel 343 into blister 344. Thesample and the C-Ab are gently mixed, illustratively by moving betweenblisters 344 and 346 via channel 345, and are incubated as describedabove. After a sufficient period of time for formation of the C-Ab-Tcomplex, the mixture is moved to blister 346 via channel 338, where amagnet 350 housed in the instrument is deployed, capturing the complexedbeads therein. The remaining fluid is moved to waste reservoir 322, viachannel 339. Optionally, pre-wash buffer from chamber 392 c is moved toblister 346 via channel 345, the magnet 350 is withdrawn, and themagnetic beads are gently washed by moving the fluid between blisters344 and 346. The magnet 350 is again deployed and the beads are againcaptured in blister 346.

Next, plunger 368 d is depressed moving the reporter antibodiesconjugated to nucleic acid template (R-Ab-DNA) to blister 346, themagnet 350 is withdrawn, and the C-Ab-T and the R-Ab-DNA are gentlymixed illustratively by moving between blisters 344 and 346 via channel345 and are incubated as described above. After formation of the ternarycomplex (C-Ab-T-R-Ab-DNA), the magnet 350 is once again deployed,capturing the ternary complex in blister 346, and the remaining fluid ismoved to waste blister 322.

The ternary complex is then washed using the wash buffer from chamber392 e, as described above for the pre-wash. The magnet 350 is againdeployed, capturing the ternary complex in blister 346, and theremaining fluid is moved to waste blister 322. Washing is repeatedvarious times, using the wash buffer from chambers 392 f through 392 k.Thus, in the illustrative embodiment of FIG. 12, seven washes arecompleted. However, as discussed above, more or fewer washes may bedesirable, depending on the particular application.

As illustrated in the Immuno-PCR II scheme shown in FIG. 11, thereporter antibody is conjugated directly to the nucleic acid template.It is understood that the reporter antibody in any of the embodimentsdiscussed herein could be attached to the nucleic acid template by anyof a variety of ways, including direct and indirect covalent andnon-covalent bonding. Also, the reporter antibody could be attached tothe nucleic acid through a variety of mechanisms, including, forexample, through the use of secondary antibodies, as illustrated in theImmuno-PCR I scheme of FIG. 11. If secondary antibodies or otherindirect coupling mechanisms are used, it may be desirable to addadditional ports and further washing steps.

The first-stage PCR master mix, as described above, is then deployed toblister 346 via activation of plunger 368 k, and the magnet 350 is onceagain withdrawn. If gentle mixing is desired, the fluid may be movedbetween blisters 346 and 364 via channel 347. While mixing can takeplace between blisters 346 and 344 as before, in the illustrativeembodiment mixing takes place between blisters 346 and 364. This aids inreducing the reintroduction of unbound reporter antibody complexes thatmay be residing in blister 344. The sample is then moved to blister 364.A bladder positioned over 364 is gently pressurized to move blister 364into contact with a heating/cooling device, such as a Peltier device,and the sample would be thermocycled, as discussed above for first-stagePCR. As discussed above in the previous example, first-stage PCR may beunnecessary with the presently described iPCR, blister 364 and itsassociated heater may be omitted, and all washes illustratively couldtake place by mixing between blisters 344 and 346. If first-stage PCR isomitted, the dilution, as discussed below may also be omitted.

Most of the amplified sample is moved to waste blister 322, leaving someamplified sample behind in blister 364 to be diluted. It is understoodthat if space constraints or other considerations limit the size ofblister 322, blisters 344 and 346 may be used to contain the remainingwaste. The small amount of remaining amplified sample is mixed withdilution buffer from chamber 392 m, which has been moved to blister 366via channel 349. The sample and the dilution buffer may be mixed gentlybetween blisters 364 and 366, via channel 355. If further dilution isdesired, dilution may be repeated using the dilution buffer from chamber392 n. Finally, some of the diluted sample is moved to waste reservoir322 and the remaining diluted sample is mixed with second-stage PCRmaster mix from chamber 392 o. After mixing, the sample is moved to thevarious low volume second stage blisters 382, where second-stage primersare provided, as discussed above. In the present configuration, blister383 may be used for a negative control and blister 384 may be used for apositive control, as discussed above in the previous iPCR example.Second-stage PCR and analysis takes place as described above in theprevious iPCR example.

Example 6 Combined PCR and iPCR

In some circumstances, it may be desirable to test for antigens andnucleic acids in one reaction set. For example, a terrorist attack mayemploy various agents to kill multiple people. In responding to theattack, it may be unknown if the causative agent is a virus, bacterium,or other organism, or if the causative agent is a toxin. Theclosed-environment system of the pouches of the present invention iswell suited for such use. In the embodiment disclosed herein, both PCRand iPCR may take place within a single pouch, allowing for simultaneousdetection of various biological and antigenic agents.

FIG. 13 shows a pouch 410 that is similar to pouch 210 of FIG. 6.Illustrative pouch 410 has all of the blisters of pouch 210, but alsoincludes blisters 430, 431, 432, and 433. Pouch 410 also has a largerfitment 490, having twenty chambers 492 with twenty correspondingplungers 468. As above, the fitment could include separate injectionports for each chamber, or various chambers could have connectingchannels. Various combinations thereof are within the scope of thisinvention. The instrument for pouch 410 would be similar to instrument800, except that additional pneumatic actuators would be needed forblisters 430, 431, 432, and 433 and channels 436, 457, 473, 486, 487,and 488, as well as two additional retractable magnets 451 and 454adjacent blisters 433 and 431, respectively.

In the illustrative embodiment, the chambers would be loaded as follows.iPCR wash buffer would be provided in chambers 492 a through 492 e and492 j. The sample to be tested would be injected into chamber 492 f. Thecapture antibodies (C-Ab) conjugated to magnetic beads are provided inchamber 492 g. An optional pre-wash buffer is provided in chamber 492 h.Reporter antibodies conjugated to their respective oligonucleotidetemplate (R-Ab-DNA) are provided in chamber 492 i. A cell lysis bufferis provided in chamber 492 k. Nucleic-acid-binding magnetic beads areprovided in chamber 492 l. Nucleic acid wash buffers are provided inchambers 492 m and 492 n. A nucleic acid elution buffer is provided inchamber 492 o. A first-stage PCR master mix is provided in chamber 492p. A dilution buffer is provided in chambers 492 q and 492 r. Asecond-stage PCR master mix is provided in chamber 492 s. Controls, asdiscussed above with respect to FIG. 6, are provided in chamber 492 t.It is understood that this arrangement is illustrative and that otherconfigurations are possible. Also, as with the other examples discussedabove, one or more of these components may be provided dried in one ormore of the blisters of pouch 410.

Once the sample is loaded into chamber 492 f and pouch 410 is loadedinto the instrument, plungers 468 f and 468 g are depressed, moving thesample and C-Ab through channel 436 to blister 430. The sample andcapture antibodies may be mixed by gently moving them between blisters430 and 431 and then incubated as described above, to encourageformation of C-Ab-T complexes. The sample is moved to blister 431 andmagnet 454 is activated, capturing the C-Ab-T complexes therein. Thus,toxins or other targeted antigens are now captured in blister 431. It isnoted that, in the illustrative embodiment, the surface of the magneticbead portion of the magnetic beads coupled to the capture antibodies isdifferent from the surface of the nucleic-acid-binding magnetic beads,and the magnetic beads coupled to the capture antibodies isillustratively configured not to bind nucleic acids. The remaining fluidis then moved to three-lobed blister 422 via channel 473. This fluid canthen be processed and assayed for the presence of target nucleic acids.This division of the sample may be problematic if a targeted antigen isa surface antigen of an organism targeted in the PCR detection. In sucha situation, it may be desirable to choose between antigen detection andnucleic acid detection for that organism, or to use separate pouches forPCR and iPCR. Alternatively, the sample may be lysed prior to antibodycapture. If lysis would interfere with antibody capture, for example bychanging the conformation of the antigen, then the sample may be dividedand just a portion of the sample may be lysed prior to antibody capture.If a pre-wash of the C-Ab-T is desired, plunger 468 h is activated andthe pre-wash buffer from chamber 492 h is moved into blister 431. Magnet454 is withdrawn, the fluid is mixed between blisters 430 and 431, andmagnet 454 is once again deployed, capturing the C-Ab-T complex inblister 431. The wash buffer, now possibly containing cells that hadbeen left behind after capture, is moved to three-lobed blister 422,along with the rest of the uncaptured material.

It is understood that the sample is now divided into two parts forseparate processing. Antigens present in the sample are now captured inC-Ab-T complexes in blister 431, while cells, viruses, and free nucleicacids present in the sample are now in three-lobed blister 422 awaitinglysis. The two portions of the sample are processed separately untilboth are ready for first-stage PCR. These processes may take place inany order or simultaneously. However, in the present embodiment, celllysis must take place prior to substantial processing of the C-Ab-Tcomplexes, so that three-lobed blister may then function as the wastereservoir. If a separate waste reservoir is used, cell lysis can bedelayed until after the C-Ab-T complexes have been processed, ifdesired.

Lysis buffer from chamber 492 k is moved into three-lobed blister 422via channel 436. Bladders adjacent the blisters of three-lobed blister422 are pressurized as described above with respect to FIG. 6, drivinghigh velocity collisions, shearing the sample, and liberating nucleicacids. Once the cells have been adequately lysed, plunger 468 l isactivated and nucleic acid binding magnetic beads stored in chamber 492l are injected via channel 436 into three-lobed blister 220. The sampleis mixed with the magnetic beads and the mixture is allowed to incubate.The processing then continues as described above with respect to thepouch of FIG. 6. The mixture of sample and beads are forced throughchannel 438 into blister 444, then through channel 443 and into blister446, where a retractable magnet 450 captures the magnetic beads from thesolution. The un-captured liquid is then forced out of blister 446 andback through blister 444 and into blister 422, which is now used as awaste receptacle. Plunger 468 m may be activated to provide a washsolution to blister 444 via channel 445, and then to blister 446 viachannel 447. Magnet 450 is retracted and the magnetic beads are washedby moving the beads back and forth from blisters 444 and 446. Once themagnetic beads are washed, the magnetic beads are recaptured in blister446 by activation of magnet 450, and the wash solution is then moved toblister 422. This process may be repeated using wash reagents inchambers 492 n. However, it is understood that more or fewer washes arewithin the scope of this invention. After washing, elution buffer storedin chamber 492 o is moved via channel 447 to blister 448, and the magnet450 is retracted. The solution is cycled between blisters 446 and 448via channel 452, breaking up the pellet of magnetic beads in blister 446and allowing the captured nucleic acids to come into solution. Themagnet 450 is once again activated, capturing the magnetic beads inblister 246, and the eluted nucleic acid solution is moved into blister448.

Returning back to blister 431, the C-Ab-T complexes are thereincaptured. Plunger 468 i is depressed and the reporter antibodiesconjugated to nucleic acid template (R-Ab-DNA) are introduced to blister430, the magnet 454 is withdrawn, and the C-Ab-T and the R-Ab-DNA aregently mixed, illustratively by moving between blisters 430 and 431 viachannel 457, and are incubated as described above. After formation ofthe ternary complex (C-Ab-T-R-Ab-DNA), magnet 454 is once againdeployed, capturing the ternary complex in blister 431, and theremaining fluid is moved to blister 422, which is now used as a wastereservoir.

The ternary complex is then washed using the wash buffer from chamber492 j, as described above for the pre-wash. Magnet 454 is againdeployed, capturing the ternary complex in blister 446, and theremaining fluid is moved to blister 422. Additional wash buffer fromchamber 492 a is injected into blister 432 via channel 486, the magnet454 is withdrawn, and the ternary complex is resuspended by mixing thefluids blisters 431 and 432. The fluids are then moved to blister 433via channel 487 and the ternary complex is captured therein viaactivation of magnet 451. The waste fluids are then moved back throughblisters 433 and 432 to blister 422. Additional wash buffer isintroduced into blister 432 from chamber 492 b and washing is repeatedby mixing between blisters 432 and 433. Washing is repeated varioustimes using the wash buffer from chambers 492 c through 492 e. Thus, inthe illustrative embodiment of FIG. 13, six washes are completed.However, as discussed above, more or fewer washes may be desirable,depending on the particular application. It is understood that blisters432 and 433 are used to minimize contamination from prior washes. Ifdesired, blisters 432 and 433 may be omitted and the wash bufferscontained in chambers 492 a through 492 e may be provided directly toeither blister 430 or 431, with mixing between blisters 430 and 431.

The washed antibody ternary complex is now captured in blister 433 andthe eluted nucleic acids are now in blister 448. It is noted that theantibody ternary complex and the eluted nucleic acids may be processedthrough PCR in independent reactions, through to separate sets ofsecond-stage PCR blisters. However, in the present embodiment theantibody ternary complex and the eluted nucleic acids are combined forPCR analysis. First-stage PCR master mix, containing all first-stageprimers, is injected from chamber 492 p into blister 448. The nucleicacid sample is then mixed between blisters 448 and 464 via channel 453.If first-stage PCR is desired for the iPCR components, the nucleic acidsample is then moved to blister 433, magnet 451 is withdrawn, and there-united sample is illustratively mixed between blisters 433 and 464.The sample is then moved to blister 464, where the sample isthermocycled, as discussed above. Next, the amplified sample may bediluted once or several times, using the dilution buffers from chambers492 q and 492 r. Prior to each dilution, a large portion of theamplified sample is removed from blister 464 via either channel 447 orchannel 488. With each addition of dilution buffer, the sample is mixedbetween blisters 464 and 466 via channel 462. After dilution, all or aportion of the sample is mixed with the second-stage PCR master mix fromchamber 492 s, as described in the examples above.

The sample is then moved from blister 466 via channel 465 to blisters482 in second-stage amplification zone 480. Blisters 482 each had beenpreviously provided with a primer pair, some of the primer pairsspecific for target nucleic acids, while other primer pairs specific foran oligonucleotide conjugated to a reporter antibody. If desired, twoblisters 482 may be dedicated to iPCR controls, as discussed above.Blisters 483 may be used for PCR controls, as discussed above withrespect to blisters 283 of FIG. 6. While 18 blisters 482 are shown, itis understood that any number of blisters 482 may be used. Second-stagePCR amplification proceeds as discussed above with respect to FIG. 6. Itis understood that PCR analysis may use amplification curves, meltingcurves, or a combination thereof, while iPCR analysis may use crossingthresholds, as discussed above. Other methods of analysis are within thescope of this invention.

Example 7: Bacterial Identification Panel

The above instruments and pouches allow for a large number of reactionsin a single, sealed environment. This is particularly true of pouch 510,which may optionally have 100 or more second-stage reaction wells 582.The large number of second-stage reactions available in pouch 510 allowsa robust identification strategy for organisms, illustratively bacteria,by allowing the interrogation of a number of genes for each species(illustratively housekeeping genes are used, as described below). Thegenes may be amplified in a first-stage multiplex reaction using outerfirst-stage primers targeting conserved sequences, and then subsequentlyamplified in individual second-stage reactions using inner second-stageprimers targeted more specifically to the individual species. Thisstrategy allows for the detection and identification of an unknown (andunspecified) pathogenic bacterium using a single set of assays performedin a closed system, with results available in real time. By combiningtests for a wide range of bacteria into a single assay, the problems aclinician faces when having to order individual assays for eachbacterial species can be reduced or avoided. This strategy may alsoreduce or eliminate the need for sequencing of the PCR amplicon fororganism identification.

One illustrative application of this method is the identification ofbacteria infecting infants in the first 90 days of life. Pediatricguidelines recommend aggressive evaluation of infants ≦28 days old forserious bacterial infection, including cultures for bacterial pathogensfrom blood, urine and, cerebral spinal fluid. While awaiting results ofthese cultures, which can take from 24-48 hours, infants are oftenhospitalized and placed on broad-spectrum antibiotics.

When an organism is identified, antibiotic susceptibility testing isoften required, and can be complex, particularly for organisms showingresistance to certain classes of antibiotics. For example, forGram-negative organisms showing resistance to third-generationcephalosporins, guidelines have been developed for detection of theextended-spectrum β-lactamase (ESBL) phenotype (35). However, from alaboratory standpoint, ESBL testing is labor-intensive (36), and from aclinical standpoint, such testing can be misleading, as sometimessusceptible isolates in vitro are resistant under treatment conditions(35-37). Delays in testing or misreporting of susceptibility data oftenresults in prolonged use of broad-spectrum agents or the risk ofinadequate therapy.

PCR-based detection of antibiotic resistance is becoming more common. Inparticular, detection of methicillin-resistance in S. aureus byidentification of the mecA gene has become widely accepted and is commonin clinical settings. However, despite the identification of thesegenetic determinants of antibiotic resistance, molecular testing forother resistance genes has not been widely used. This likely is due tothe complexity of the genetics. For example, over 200 ESBL genotypeshave been characterized, reflecting multiple mutations in seven or moreplasmid-encoded genes. Diagnostic strategies such as those illustratedhere, capable of high-order multiplex analysis and the flexibility tochange as the resistance landscape evolves, will aid in the developmentof rapid testing for these resistance determinants

Currently, molecular testing is not routinely available for any of thebacterial pathogens common in young infants. Even limiting the list ofbacteria tested to that of those most likely found in these infants,this list would include 8-10 organisms, and if molecular testing wereavailable, traditional PCR methods would require a minimum of 8-10separate tests. Alternately, using currently described broad-range PCRstrategies, a sequencing step, taking almost as long as culture, wouldbe necessary for identification. A strategy such as that presented inthis Example could allow for interrogation of blood or CSF or othersterile body fluid for multiple pathogens in a single assay with resultspotentially available at the point-of-care. By way of example, 27pathogens (4 genes*27 pathogens=108 wells+12 control wells=120 totalsecond-stage reaction wells 582 in the high-density array) may be testedin a single pouch 510.

It is known that different groups of patients have different likelihoodsof infection with particular organisms based on their age, presentinglocation (for example, inside or outside the hospital), and underlyingimmunity. By designing broad-range outer primer sets for conserved genesfor first-stage PCR, the present strategy allows for the modification ofsecond-stage inner primer sets to target different populations ofpatients. In the example described above with respect to infants, innersecond-stage PCR targets illustratively would include (but not belimited to) Neisseria meningitidis, Haemophilus influenzae, Escherichiacoli, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcusagalactiae, Staphylococcus aureus and Listeria monocytogenes. It isunderstood that this list is exemplary only and that the list ofsecond-stage targets may be adjusted due to a wide variety of factors.Other populations that could be targeted include: patients in theintensive care unit, (ICU panel illustratively including Pseudomonasaeruginosa, Enterobacter cloacae, Klebsiella oxytoca and Serratiamarscescens), and patients with malignancy and immunity suppressed bychemotherapy (illustratively febrile neutropenic panel, includingopportunistic pathogens such as Staphylococcus epidermidis, Pseudomonasaeruginosa, and Viridans group Streptococci). Illustratively, in eachcase the optimized first-stage outer primer set may remain unchanged,but the panel of second-stage inner primers would be modified for thetarget population.

This strategy could also be used for identification of organisms fromculture, particularly in cases where a patient is not responding toantibiotics, and rapid organism identification might direct antibiotictherapy more appropriately, or in situations, such as possible infectionwith Neisseria meningitidis (meningococcemia) where rapid speciesconfirmation is crucial to the initiation of prophylaxis for contacts.

In addition, it is possible that this strategy may allow theidentification of novel strains of bacteria causing disease byidentifying bacteria based on a Cp and/or post-second-stageamplification melting curve “fingerprint.” Due to the broad-range designof first-stage outer primers, which are likely to amplify targets fromeven unknown bacteria, along with expected cross-reactivity of innersecond-stage primers with non-target organisms, a novel bacterium mayhave a “fingerprint” not previously encountered. It is also possiblethat different strains of the same species of bacteria might generateunique “fingerprints” (either by Cp pattern or post-second-stageamplification melting curves) and thus this technique may allowepidemiologic tracing of bacteria and outbreaks of infection. Currently,multi locus sequence typing (MLST) can be used for this purpose, butthis technique takes a significant period of time to perform. Thepresent strategy would allow for rapid identification of an outbreak,illustratively by coding instrument 800 to detect when unique Cppatterns are seen at a high frequency over a short period of time.

Theoretical Support for the Strategy.

With the advent of genomic sequencing, systematic bacteriology hasworked to come up with a definition of “species” based on genomicsequencing (Gupta. Micro and Mol Biol Rev. 1998; 62: 1435, Gupta et al.Theoretical Pop Biol. 2002; 61: 423). The “Report of the Ad HocCommittee for the Re-Evaluation of the Species Definition inBacteriology” (Stackebrandt et al. Int J Systemic and Evol Microbiol.2002; 52: 1043) recommends evaluation of “protein-coding genes understabilizing selection” (housekeeping genes) for their use inphylogenetic studies to differentiate bacterial species from oneanother. The present strategy uses similar genes applied to theidentification of known and well-described pathogenic species,illustratively with results in real-time. The use of the particulargenes in the present example is further supported by other studies inwhich the utility of a range of housekeeping genes in identifying andclassifying species has been compared to previously accepted methodssuch as DNA:DNA reassociation, absolute nucleotide identity and 16S rRNAgene sequencing (Stackebrandt et al. Int J Systemic and Evol Microbiol.2002; 52: 1043, Konstantinos et al. Appl Environ Micro. 2006; 72: 7286(multiple genes), Mollet et al. Mol Micro. 1997; 26: 1005 (rpoB), Kwoket al Int J Systemic and Evol Microbiol. 1999; 49: 1181 (hsp60)).Although the present strategy bases gene selection on these types ofstudies, it is understood that other genes may be used as well.

Primer Design.

The illustrative strategy employs broad-range outer first-stage PCRprimers targeting multiple conserved protein-encoding genes andconserved non-protein-encoding sequences such as ribosomal genes.Identification of specific bacterial species is done in individualsecond-stage wells 582 of a 120 well high-density array, using specificinner second-stage PCR primers for pathogens of interest. Theillustrative broad-range outer first-stage primer sets amplify conservedgenes in phylogenetically distant bacterial pathogens. It is anticipatedthat 4-7 genes may need to be interrogated to generate a robust andspecific method for bacterial identification; it is possible that,despite directing the assays at conserved genes, a particular strain orisolate may undergo an unanticipated nucleotide sequence change in theregion of one assay, which could be rescued by a positive assay foranother gene. Illustrative initial gene targets include rpoB (RNApolymerase beta subunit), gyrB (DNA gyrase subunit B), ftsH (conservedmembrane protease), ompA (outer membrane protein A), secY (conservedmembrane translocon), recA (DNA recombination protein), groEL (hsp60heat shock protein) and the 16S rRNA gene. Primer design and initialtesting is presently underway for rpoB and gyrB.

Due to the fact that the target species come from distant arms of thebacterial phylogenetic tree, the ability to perform simple nucleotidealignment to interrogate for conserved regions is unlikely. By aligningthe genes based on their amino acid sequences, it is possible toidentify protein domains conserved across species, and it is to thenucleotide sequences of these domains that the outer first-stage primersof the nested assay are designed. By using degenerate primers, it hasbeen found that rpoB and gyrB require only 2 outer first-stage primersets (one for Gram-positive and one for Gram-negative organisms; 2-4primers per set, although it is understood that other numbers of primersets or primers per set could be used, as desired for the particularapplication) per gene to amplify all target organisms tested (14 speciesto date). It is possible that other gene targets will require a largernumber of outer first-stage primers. By limiting the number offirst-stage primer sets in the outer reaction, the multiplex of thefirst-stage PCR amplification is simplified, thus increasing the chancesof a successful assay. Further, the outer multiplex, which is often moresensitive to changes in primer number and sequence, can remainundisturbed if the panel changes, and does not need to be re-validatedfor all pathogens if one or more pathogen is added or removed. While itis desired to have a single set of first-stage primers and only changethe second-stage primers for specific panels, it is also possible thatsome panels may require unique first-stage primer sets. In the presentexample, degeneracies in the illustrative outer first-stage primer setsfor rpoB and gyrB range from 6 to 32 fold.

Illustratively, desired locations for outer first-stage primers arelocations in which two conserved regions, separated by no more thanabout 500 nucleotide base pairs, bracket a variable region with enoughsequence variability to design inner primers specific to particularbacterial species. Such locations were identified in both rpoB and gyrB,and multiple inner primer sets have been designed to the variableregions, each, for the most part, targeting a specific bacterialspecies. Where possible, the 3′-end of the primer has been designed tolie on a sequence that encodes a “signature” amino acid or acids whichis unique to the species being targeted, but different from otherbacteria. This diminishes or prevents second-stage amplification unlessthat species is present. This was not possible in all cases, and somecross-amplification is seen. Illustrative primers developed to date forrpoB and gyrB are shown in Tables 1-3.

When bacterial species are genetically too similar to be amplifiedexclusively by a unique set of primers, a “fingerprinting” techniquebased on the amplification characteristics of a set of primers can beused for species identification. An illustrative example is “enteric”organisms (e.g. E. coli, K pneumoniae, Koxytoca and E. cloacae) in therpoB gene. These enteric organisms have too much sequence similaritywithin rpoB, and designing specific inner second-stage primers hasproven difficult. Therefore, a “pan-enteric” inner second-stage primerwas designed, as well as “preferential” primers, as shown in Table 3. Anexample of how these primers would be used to generate a fingerprint isshown in FIGS. 22-23. When the preferential inner primers are used, eachenteric bacterial template shows a different pattern of crossing points,and each organism amplifies best with its own preferential inner primerset. FIG. 22a shows second-stage amplification with the pan-entericprimers. All three organisms amplified with essentially the same Cp.FIGS. 22b-d show amplification using the preferential primers for E.coli, K pneumoniae, and K. oxytoca and E. cloacae, respectively. In thisillustrative example, the inner primers for K. pneumoniae have the mostsequence difference, and, thus, are the most selective, with K.pneumoniae having a much earlier Cp (FIG. 22c ). FIG. 23a-b show a chart(FIG. 23a ) and decision tree (FIG. 23b ) that may be used foridentification using the Cp fingerprint. As illustrated in Table 3, thebases in bold provide primer specificity. In this illustrative example,the specificity is provided by the forward primers; the reverse primerfor the E. coli, K. pneumoniae and E. cloacae assays is the same.

Enteric organisms can be differentiated from each other in the gyrBassay, as well as potentially by the melting temperature of the rpoBamplicon, depending on sequence variation. An additional strategy fordistinguishing closely-related species, for example the entericorganisms already discussed, would be to target a gene which is presentwithin that group, but less-conserved than other housekeeping genes, andnot present in other groups of bacteria. Illustratively, an outermembrane protein gene such as ompA (outer membrane protein A), presentin the outer membrane of Gram-negative bacteria, but not present inGram-positive bacteria, could be used. Outer primers would targetregions conserved within the closely-related group of organisms, andinner primers would be used to distinguish them. An illustrative set ofprimers for ompA is shown in Table 4. Thus, it is understood thatvarious combinations of first- and second-stage reactions can be used,including those identifying a particular group of organisms and othersspecific for a particular target species, for example specific bacterialspecies.

While the rpoB (RNA polymerase beta subunit), gyrB (DNA gyrase subunitB), and ompA (outer membrane protein A) genes are used in this example,it is understood that other genes may be used as well, including ftsH(conserved membrane protease), secY (conserved membrane proteintranslocon), recA (DNA recombination protein), groEL (hsp60 heat shockprotein) and the 16S rRNA gene. It is understood that these genes areillustrative only, and any combination of these and/or other genes iswithin the scope of this invention. While variability in the targetsequence at the 3′-end may be used to limit cross-amplification, it isanticipated that cross-amplification by the inner primers will occur,possibly in several genes even for the same species. This may lead to a“positive call” for more than one organism-specific assay.Identification of each organism may be made by its “fingerprint” orpattern of amplification (whether amplification occurs and Cp) over allinner primer sets.

TABLE 1 Outer First-Stage Primers rpoB and gyrB Gene Primer SequencerpoB forward GGCTTYGARGTDCGDGACG Gram-negatives (SEQ ID NO. 1) rpoBreverse RCCCATBARTGCRCGGTT Gram-negatives (SEQ ID NO. 2) rpoB forwardBCACTAYGGBCGYATGTGTCC Gram-positives (SEQ ID NO. 3) rpoB reverseAAHGGAATACATGCYGTYGC Gram-positives (SEQ ID NO. 4) gyrB forwardTCCGGYGGYYTRCAYGG Gram-negatives-1 (SEQ ID NO. 5) gyrB forwardTCBGTNGTWAACGCCCTGTC Gram-negatives-2 (SEQ ID NO. 6) gyrB reverseRCGYTGHGGRATGTTRTTGGT Gram-negatives (SEQ ID NO. 7) gyrB forwardGGYGGWGGYGGMTAYAAGGTTTC Gram-positives-1 (SEQ ID NO. 8) gyrB forwardGGMGGYGGCGGMTAYAAAGTATC Gram-positives-2 (SEQ ID NO. 9) gyrB reverseTTCRTGMGTHCCDCCTTC Gram-positives-1 (SEQ ID NO. 84) gyrB reverseTTCATGCGTACCACCCTC Gram-positives-2 (SEQ ID NO. 10)

TABLE 2 Inner Second-Stage Primers: rpoB and gyrB Gene Primer NameSequence Target rpoB rpoB1Ents.iF04 CCGTAGYAAAGGCGAATCC Enteric(SEQ ID NO. 11) organisms rpoB rpoB1Ents.iR03 GGTWACGTCCATGTAGTCAACEnteric (SEQ ID NO. 12) organisms rpoB rpoB1Hinf.iF01ACTTTCTGCTTTTGCACGT H. influenzae (SEQ ID NO. 13) rpoB rpoB1Hinf.iR01CTTCAGTAACTTGACCATCAAC H. influenzae (SEQ ID NO. 14) rpoB rpoB1Nmen.iF01CTCATTGTCCGTTTATGCG N. meningitidis (SEQ ID NO. 15) rpoB rpoB1Nmen.iR02TCGATTTCCTCGGTTACTTTG N. meningitidis (SEQ ID NO. 16) rpoBrpoB1Paer.iF01 TCAACTCCCTGGCGACC P. aeruginosa (SEQ ID NO. 17) rpoBrpoB1Paer.iR01 CYACGCGGTACGGGC P. aeruginosa (SEQ ID NO. 18) rpoBrpoB1Spne.iF03 AGGTTGACCGTGAAACAGG S. pneumoniae (SEQ ID NO. 19) rpoBrpoB1Spne.iF04 AGGTTGACCGTGCAACTGG S. pneumoniae (SEQ ID NO. 20) rpoBrpoB1Spne.iR03 GCTGGATACTCTTGGTTGACC S. pneumoniae (SEQ ID NO. 21) rpoBrpoB1Spne.iR04 GCTGGATATTCTTGGTTAACC S. pneumoniae (SEQ ID NO. 22) rpoBrpoB1Spyo.iF01 YGAAGAAGACGARTACACAGTT S. pyogenes (SEQ ID NO. 23) rpoBrpoB1Spyo.iR01 CGTCAACGAAATCAACAACAC S. pyogenes (SEQ ID NO. 24) rpoBrpoB1Saga.iF01 TGAAGAAGATGAATTTACAGTT S. agalactiae (SEQ ID NO. 25) rpoBrpoB1Saga.iR01 CGTCAACAAAGTCAACAATGC S. agalactiae (SEQ ID NO. 26) rpoBrpoB1Saur.iF02 GTAAAGTTGATTTAGATACACATGC S. aureus (SEQ ID NO. 27) rpoBrpoB1Saur.iR01 CGACATACAACTTCATCATCCAT S. aureus (SEQ ID NO. 28) rpoBrpoB1Sepi.iF01 YTWGATGAAAATGGTCGTTTCYTAG S. epidermidis (SEQ ID NO. 29)rpoB rpoB1Sepi.iR01 GTTTWGGWGATACRTCCATGT S. epidermidis (SEQ ID NO. 30)rpoB rpoB1Lmon.iF01 CKAACTCGAAATTAGACGAACAA L. (SEQ ID NO. 31)monocytogenes rpoB rpoB1Lmon.iR01 TTTTCTACCGCTAAGTTTTCTGA L.(SEQ ID NO. 32) monocytogenes rpoB rpoB1Efas.iF01 ACAGCYGAYATCGAAGACCAE. faecalis (SEQ ID NO. 33) rpoB rpoB1Efas.iR01 ACTTCTAAGTTTTCACTTTGHGCE. faecalis (SEQ ID NO. 34) rpoB rpoB1Efas.iR02 ACTTCTAAGTTTTCACTTTGTAGE. faecalis (SEQ ID NO. 35) gyrB gyrB1Hinf.iF01 ATTCGTCGTCAAGGTCACH. influenzae (SEQ ID NO. 36) gyrB gyrB1Hinf.iR01 CGATTGAGGCTCCCCTAAAH. influenzae (SEQ ID NO. 37) gyrB gyrB1Ecol.iF01 GTTATCCAGCGCGAGGGTAE. coli (SEQ ID NO. 38) gyrB gyrB1Ecol.iR01 GTGCCGGTTTTTTCAGTCT E. coli(SEQ ID NO. 39) gyrB gyrB1Kpne.iF01 GATAACAAAGTTCACAAGCAGATGK. pneumoniae (SEQ ID NO. 40) gyrB gyrB1Kpne.iR01 GTCGGTTTCGCCAGTCAK. pneumoniae (SEQ ID NO. 41) gyrB gyrB1Koxy.iF02 GTTTCTGGCCGAGCTAYGAK. oxytoca (SEQ ID NO. 42) gyrB gyrB1Koxy.iR02 CGTTTTGCCAGRATYTCGTATK. oxytoca (SEQ ID NO. 43) gyrB gyrB1EcloiF01 TATCCAGCGCGAAGGCAE. cloacae (SEQ ID NO. 44) gyrB gyrB1Eclo.iR01 GGCCAGAAACGCACCATE. cloacae (SEQ ID NO. 45) gyrB gyrB1Nmen.iF01 AACACTTCGTCCGCTTCGTN. meningitidis (SEQ ID NO. 46) gyrB gyrB1Nmen.iR01 GCGAGGAAGCGCACGGTN. meningitidis (SEQ ID NO. 47) gyrB gyrB1Paer.iF01 TCGCCACAACAAGGTCTGP. aeruginosa (SEQ ID NO. 48) gyrB gyrB1Paer.iR01 GGCCAGGATGTCCCARCTP. aeruginosa (SEQ ID NO. 49) gyrB gyrB1Saur.iF01GTCATTCGTTTTAAAGCAGATGGA S. aureus (SEQ ID NO. 50) gyrB gyrB1Saur.iR01GTGATAGGAGTCTTCTCTAACGT S. aureus (SEQ ID NO. 51) gyrB gyrB1Spne.iF01AGAATACCGTCGTGGTCAT S. pneumoniae (SEQ ID NO. 52) gyrB gyrB1Spne.iR01ACCTTGGCGCTTATCTGT S. pneumoniae (SEQ ID NO. 53) gyrB gyrB1Spyo.iF01AACGACTCAGTTTGATTACAGTG S. pyogenes (SEQ ID NO. 54) gyrB gyrB1Spyo.iR01AAATGTTCTTCTTGTTCCATACCT S. pyogenes (SEQ ID NO. 55) gyrB gyrB1Saga.iF01GYAAGGTTCATTATCAAGAATAYCAA S. agalactiae (SEQ ID NO. 56) gyrBgyrB1Saga.iR01 AAGTGAACTGTTGTTCCTGATAAG S. agalactiae (SEQ ID NO. 57)gyrB gyrB1Sepi.iF01 GCTATTCGATTCAAAGCCGATAA S. epidermidis(SEQ ID NO. 58) gyrB gyrB1Sepi.iR01 TCTAACTTCCTCTTCTCTTTCATCTTTS. epidermidis (SEQ ID NO. 59) gyrB gyrB1Efas.iF01CGTCGTGAAGGACAAGAAGATAAA E. faecalis (SEQ ID NO. 60) gyrB gyrB1Efas.iR01CTTGTTGCTCTCCTTCAATG E. faecalis (SEQ ID NO. 61) gyrB gyrB1Lmon.iF03CCAATTTATTTGGAAGGTGAACG L. (SEQ ID NO. 62) monocytogenes gyrBgyrB1Lmon.iR05 GCGAATGAAATGATRTTGCTTGAGA L. (SEQ ID NO. 63)monocytogenes

TABLE 3 Inner Primers for Enteric Organisms in rpoB Preferential TargetPrimer Sequence “pan- rpoB1Ents.iF04 CCGTAGYAAAGGCGAATCC enteric”(SEQ ID NO. 11) rpoB1Ents.iR03 GGTWACGTCCATGTAGTCAAC (SEQ ID NO. 12)E. coli rpoB1Ecol.iF01 ACTCCAACCTGGATGA A GA (SEQ ID NO. 64)rpoB1Ents.iR02 ACGTCCATGTAGTCAACC (SEQ ID NO. 65) K. rpoB1Kpne.iF01GAACTCCAACCTGGATGAAA A C pneumoniae (SEQ ID NO. 66) rpoB1Ents.iR02ACGTCCATGTAGTCAACC (SEQ ID NO. 65) E. cloacae/ rpoB1Eclo.iF01ACTCCAACCTGGATGA C GA K. oxytoca (SEQ ID NO. 68) rpoB1Ents.iR02ACGTCCATGTAGTCAACC (SEQ ID NO. 65)

TABLE 4 Outer and Inner Primers for Enteric Organisms in ompA TargetPrimer Sequence Outer primers Enterics ompA1Ents.oF_aTCGCYACCCGTCTGGAATA (SEQ ID NO. 70) ompA1Ents.oR_a CGTCTTTMGGRTCCAKGTTG(SEQ ID NO. 71) Inner primers E. coli ompA1Ecol.iF_aGTCTGGAATACCAGTGGACC (SEQ ID NO. 72) ompA1Ecol.iR_a CTGATCCAGAGCAGCCT(SEQ ID NO. 73) K. ompA1Kpne.iF_a CGGTCAGGAAGATGCTGC pneumoniae(SEQ ID NO. 74) ompA1Kpne.iR_a GGTGAAGTGCTTGGTAGCC (SEQ ID NO. 75)K. oxytoca ompA1Koxy.iF_a CGGTCAGGAAGATGTTGCT (SEQ ID NO. 76)ompA1Koxy.iR_a TGACCTTCTGGTTTCAGAGTAGAT (SEQ ID NO. 77) E. cloacaeompA1Eclo.iF_a AACATCGGCGACGGCAA (SEQ ID NO. 78) ompA1Eclo.iR_aGCTGGAGCAACGATTGGC (SEQ ID NO. 79)

As discussed above, while ideally each organism would be amplifiedexclusively by its appropriate inner second-stage primer set, crossamplification can occur. Due to expectations that organisms will showmultiple, but predictable, amplification curves in a single conservedgene (see the example with K. oxytoca and H. influenzae rpoB primers inFIG. 18 described below), use of supervised learning is anticipated forthe instrument 800 software, based on the pattern of amplificationcurves generated by the panel of conserved gene assays. For eachbacterial species, clinical isolates whose identity has been confirmedby biochemical identification and, where appropriate, gene specific DNAsequencing, will form the basis for training data set for thepredication software. Depending on the characteristics of the data, itis anticipated that either a nearest-neighbor algorithm or a PrincipleComponent Analysis based ranking system will be used to generate anidentity “fingerprint” for each target bacteria.

Given the complexity of a nested multiplex amplification reaction,considerable optimization may be required. In one illustrative method,outer and inner primer sets are initially tested separately in simplePCR reactions (one primer set, one template) to demonstrate whether eachprimer set will generate a template-dependent real-time amplificationcurve, with a resultant PCR product of the expected size.Illustratively, size may be demonstrated by agarose gel electrophoresis,although other methods are known in the art and are within the scope ofthis disclosure. Further assay optimization illustratively takes placeusing nested PCR and real-time detection (for example, amplifiedproducts may be detected using a fluorescent double-stranded DNA bindingdye).

The fluorescence crossing point (Cp), of the nested reaction providesrelative quantitation of the initial target, and can also be used as ameasure of the efficiency of the PCR reactions. Comparison of Cps allowsevaluation of assays run under different conditions. Both inner andouter primer sets may be optimized to generate early nested Cps,representing efficient PCR. Other parameters examined in optimizationmay be the Cp of the reaction when no template is added and the Cp ofthe reaction when human genomic DNA is used as a template. Productsformed in the no-template reaction are non-specific and likely representprimer dimers. Products in the human genomic reaction that are notpresent in the no-template reaction represent cross-reaction with humansequences, and may interfere with specificity when the assay isperformed directly from clinical specimens. In both cases, theamplification curves are false-positives, and primer sets with Cps of,illustratively, less than 32 cycles in these reactions are redesigned.While 32 cycles is chosen as the Cp cut-off point, it is understood thatother crossing points could be chosen, depending on the desiredspecificity and sensitivity.

In the illustrative example, once assays have been optimized insingleplex format, assays may then be multiplexed and tested for limitof detection and specificity (amplification of only the intendedtarget), with comparison to the singleplex assays. Assays may beredesigned as necessary. During multiplex optimization, assays may bemoved to instrument 800, so that optimization may occur under theconditions of the final assay.

Two sets of experiments demonstrating the illustrative bacterialidentification strategy are shown. FIG. 18 shows an experiment in whichboth rpoB and gyrB are used for identification and differentiation ofGram-negative organisms. Bacteria from different phyla have beenincluded to help display the broad range of the outer first-stageprimers, and because they are important antibiotic-resistantGram-negative pathogens that could be targeted in an illustrative assayaccording to the methods discussed herein. In the experiment shown inFIG. 18, DNA was extracted from clinical isolates of the selectedorganisms. Total bacterial nucleic acid was amplified in a multiplexouter first-stage PCR reaction containing primer sets targeting both therpoB and gyrB genes of Gram-negative organisms (one degenerate primerpair per gene). All first-stage amplicons were then nested into allinner second-stage PCR reactions each containing a primer set specificfor a particular organism and amplified in the presence of LC Green®Plus, although other detection means may be used, as described above. Asshown in FIG. 18, although first-stage amplicons for each bacterium aretested with all inner second-stage primer sets, the only inner primersets showing significant amplification are those directed at the targetorganism. This demonstrates the specificity of the inner second-stageassay. FIG. 19 shows an experiment looking at the rpoB gene, in thiscase with Gram-positive targets. As was seen in FIGS. 18A-C, the onlyinner second-stage primer set showing significant amplification is thatdirected at the target organism. FIG. 20 shows amplicon melting profilesfor the second-stage amplicons generated in the experiment shown in FIG.18. As can be seen in FIG. 20, the amplicons melt with characteristicprofiles, which could be used for further specificity in organismidentification. Subsequently, second stage primers have been developedfor S. epidermidis, K. oxytoca, and E. faecalis for use with the samefirst-stage primers.

The above examples were done as individual reactions, for the purposesof optimizing. FIGS. 21a-b show amplification with a first-stageamplification and second-stage amplification done in an instrument 800.Inner second-stage primers were spotted in triplicate in second-stage581. The replicates were spotted in locations remote from each other, tominimize positional effects. The results shown in FIG. 21a are for theN. meningitidis gyrB assay. The three amplification curves with thelowest Cp (about 18-20) are the neisseria amplification curves, whilethose coming up later (Cp>25) show primer-dimers or other non-specificamplification. FIG. 21b shows the results for the N. meningitidis rpoBassay, confirming the results from the gyrB gene, with even greaterseparation between the target amplification and the non-specificamplification. In addition to Cp, the results may be confirmed usingmelting curve analysis. Similar results have been obtained with othertargets. Clinical samples have been run on this panel, resulting in 0-4positive targets per sample.

It is understood that while the above examples are used for identifyingbacterial species, the method could be applicable to identification ofunknown bacteria. Illustratively, second stage inner primers could bedesigned more generally, and spread throughout the “sequence space” ofthe variable inner region. Bacterial identification would then be basedon the Cp “fingerprint” across the second stage PCR array. The bacterialtraining set described above would be used to determine fingerprints forknown bacterial species, and additional species/novel fingerprintsencountered over time could be added to the data.

Additionally, while the above examples focus on identifying bacterialspecies, a similar method could be used to identify antibioticresistance, with or without species identification. Many antibioticresistance genes are found on plasmids, and for treatment purposes, thepresence or absence of the gene or gene mutation may be more importantthan identification of the pathogen. As an illustrative example, onecould test for one or more of the TEM, SHV, and CTX-M β-lactamase genesfound on plasmids in Gram-negative enteric pathogens. In this example,outer primers would target conserved portions of the plasmid; innerprimers could also target conserved regions when the presence or absenceof the plasmid confers resistance (e.g. ampicillin resistance), oralternatively could target regions in which mutations conferringdifferent resistance properties (e.g. the extended-spectrum β-lactamasephenotype) are found. If point mutations lead to a change in theresistance pattern, probes can be included in the second stage reactionto identify the mutation, illustratively by probe melting. The probesmay be labeled, or the second-stage reaction may include a dsDNA bindingdye, illustratively a saturation dye (see, e.g., U.S. patent applicationSer. No. 11/485,851, herein incorporated by reference). Table 5 showsillustrative probes targeting several mutations in the TEM β-lactamasegene.

WT Mutant Probe codon codon TEM (104) ACTTGGTTgAGTACTCACCAGTCACAGAA GAGwt (SEQ ID NO. 80) TEM (104) ACTTGGTTaAGTACTCACCAGTCACAGAA GAG aAG EK(SEQ ID NO. 81) TEM (164) CCTTGATcGTTGGGAACCGGAGCTGAAT CGT wt(SEQ ID NO. 82) TEM (164) CCTTGATaGTTGGGAACCGGAGCTGAAT CGT aGT RS(SEQ ID NO. 83)

Further, it is understood that the methods described herein could beused for the identification of other, non-bacterial species. Asdescribed above, the outer first-stage primers may be targeted togenerally conserved regions of target genes, while the innersection-stage primers would be targeted to sequences specific for thespecies or genus. In one example, the methods described herein could beapplied to fungal identification. Such an application of the presentinvention could significantly reduce the time to diagnosis, assporulation, which can take several days to several weeks, is currentlyused to identify a fungal species. In some cases, a fungus may notsporulate at all in the clinical laboratory, and would only beidentifiable by molecular methods. If four conserved genes are requiredfor the outer first-stage amplification, twenty-five or more innersecond-stage targets may be tested per gene, assuming approximately 100second-stage wells 592. Other non-limiting potential combinations are asfollows:

8 genes×12 inner primers=96 reactions

7 genes×14 inner primers=98 reactions

6 genes×16 inner primers=96 reactions

5 genes×20 inner primers=100 reactions

It is understood that these combinations are illustrative only, and thatother combinations are possible. For example, if there are more secondstage wells 592 in high density array 581, then additional genes and/ormore inner primer sets may be used. If desired, amplicon meltingtemperatures and/or probes may be used for further identification or forconfirmation of results.

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While references are made herein to PCR and iPCR, it is understood thatthe devices and methods disclosed herein may be suitable for use withother nucleic acid amplification or other biological processing methods,as are known in the art, particularly methods that benefit from afirst-stage multiplex reaction and a second-stage individual reaction.Illustrative non-limiting second-stage reactions include primerextension, including allele-specific primer extension; extensionterminations, including termination by incorporation of one or moredideoxy nucleotides; incorporation of fluorescent or non-fluorescentlabels; and other enzymatic reactions requiring a change in reactionmixture components or component ratios, such as asymmetric PCR,allele-specific PCR, invader assays, and other isothermal amplificationor detection chemistries.

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

1-31. (canceled)
 32. A method for identifying a bacterial speciescomprising the steps of obtaining a sample containing the bacterialspecies, amplifying, in a single reaction mixture containing nucleicacid from the sample, a plurality of bacterial genes using pairs ofouter first-stage primers designed to hybridize to generally conservedregions of the bacterial genes to generate a plurality of first-stageamplicons, dividing the reaction mixture into a plurality ofsecond-stage reactions, subjecting each of a plurality of thesecond-stage reactions to amplification conditions, wherein each of theplurality of second-stage reactions uses a pair of second-stage primers,each pair of second-stage primers specific for a different targetbacterial species or group of bacterial species, generating anamplification curve for each of the second-stage reactions thatamplified, determining a crossing point for each amplification curve,and identifying the bacterial species based on an order of a pluralityof the crossing points.
 33. The method of claim 32, wherein each of thefirst-stage amplicons is subjected to amplifying conditions with eachpair of second-stage primers, each pair of second-stage primers inseparate second-stage reactions.
 34. The method of claim 32, wherein anearly crossing point in one of the second-stage reactions indicatesspecificity between one of the first-stage amplicons and its respectivepair of second-stage primers and a late crossing point in another one ofthe second-stage reactions indicates some lack of specificity betweenthe one of the first-stage amplicons and its respective pair ofsecond-stage primers.
 35. The method of claim 34, wherein each pair ofsecond-stage primers is unique.
 36. The method of claim 34, whereinseveral of the second-stage reactions have a first member of the pair ofsecond-stage primers in common, and a second member of the pair ofsecond-stage primers that have sequence differences, wherein thesequence differences provide the amount of specificity.
 37. The methodof claim 32, wherein the identifying step further comprises performingmelting curve analysis on the amplified second-stage reactions to obtaina Tm for each of the amplified second-stage reactions, to confirm orfurther identify the bacterial species.
 38. The method of claim 32,wherein a plurality of the second-stage primers each have a 3′-endsequence for its specific target species.
 39. The method of claim 32,wherein the second-stage primers are nested internally with respect tothe first-stage primers.
 40. The method of claim 32, wherein theamplifying step comprises amplifying at least one antibiotic resistancegene, at least one of the plurality of second-stage reactions comprisessecond-stage primers specific for the antibiotic resistance gene, andsubsequent to the subjecting step, the method further comprises the stepof identifying whether amplification of the antibiotic resistance geneoccurred.
 41. The method of claim 40, wherein the second-stage reactionthat comprises the second-stage primers specific for the antibioticresistance gene further comprises a probe, and the method furthercomprises identifying whether a gene mutation is present in theantibiotic resistance gene by melting of the probe.
 42. A method foridentifying an organism comprising the steps of amplifying, in a singlereaction mixture containing nucleic acid from the organism, a pluralityof conserved genes using outer first-stage primers designed to hybridizeto generally conserved regions of the conserved genes to generate aplurality of first-stage amplicons, dividing the reaction mixture into aplurality of second-stage reactions, subjecting each of a plurality ofthe second-stage reactions to amplification conditions, wherein each ofthe plurality of the second-stage reactions uses a pair of second-stageprimers, each pair of second-stage primers specific for a differentorganism or genus of organisms, generating an amplification curve foreach of the second-stage reactions that amplified, determining acrossing point for each amplification curve, and identifying theorganism based on an order of a plurality of the crossing points. 43.The method of claim 42 wherein the organism is a bacterium.
 44. A methodfor identifying an antibiotic resistance gene in a sample comprising thesteps of amplifying, in a single reaction mixture containing nucleicacid from the organism, at least one of a plurality of respectiveantibiotic resistance genes using outer first-stage primers designed tohybridize to regions of the respective genes to generate a plurality offirst-stage amplicons, dividing the reaction mixture into a plurality ofsecond-stage reactions, subjecting each of the plurality of thesecond-stage reactions to amplification conditions, wherein each of theplurality of second-stage reactions uses a pair of second-stage primers,each pair of second-stage primers specific for a different one of theplurality of respective antibiotic resistance genes, generating anamplification curve for each of the second-stage reactions thatamplified, determining a crossing point for each amplification curve,and identifying the antibiotic resistance gene based on an order of aplurality of the crossing points.
 45. The method of claim 44 wherein aportion of the second-stage primers are specific for regions of therespective antibiotic resistance genes in which mutations conferringdifferent resistance properties are found.
 46. The method of claim 44wherein a portion of the second-stage reactions each further comprise aprobe targeting a mutation, which is a mutation that leads to a changein antibiotic resistance.
 47. The method of claim 46 wherein theidentifying step also includes determining melting curve temperature ofthe probe.